The Algicidal Potential of Some Volatile Substances on Oil Base: Effect of Structure–Species–Effectivity Relationships

Abstract

The bio-colonization of building materials by green algae is a widespread problem. To prevent this, it is advisable to use natural substances to avoid environmental damage. This study examined the effectiveness of four essential oils (cinnamon, thyme, oregano and hop) and four oil-based substances (trans-cinnamaldehyde, thymol, carvacrol and β-caryophyllene) in preventing bio-colonization. The effectiveness of these chemicals against three algal species (Haematococcus pluvialis, Chlorella mirabilis and Stichococcus sp.) and a mixture of these species was tested. The tests were carried out under laboratory conditions over a period of 14 days. The concentrations tested were in the range of 3–200 mg/L. Growth densities were assessed spectrometrically as absorbencies at a wavelength of 750 nm. Caryophyllene, thymol, oregano oil, and hop oil did not negatively affect the growth of algal biomass. The algicidal effect increased in the following order for the other chemicals: cinnamon oil and trans-cinnamon aldehyde < thyme oil and carvacrol. Their biocidal effect was influenced by their structure, particularly their molecular weight and solubility in fat (log K_ow). H. pluvialis was a less sensitive species than the smaller S. sp and Ch. mirabilis. The artificial biofilm was sensitive to thyme oil and carvacrol, similarly to natural biofilms, as was demonstrated in previously published studies.

1. Introduction

Green algae are among the most widespread groups of organisms in the world. They inhabit various aquatic and terrestrial environments [1,2]. These photoautotrophic organisms play a key role in oxygen production, but they can also have harmful effects. They are often found on building stones, where they form biofilms together with other organisms [3,4]. Consequently, they play a significant role in the biodeterioration of building materials. They can metabolize various organic acids and compounds that etch the surface of building materials [5,6,7,8,9]. Their decomposition produces simpler compounds that can serve as a nutrient source for other microorganisms, thereby supporting the systematic deterioration of surfaces [10].

Algae can attack all known building materials. Most studies focus on the restoration of historical buildings, paintings, plaster, marble, and wooden structures [11,12]. Algae inhabit outdoor materials due to their phototrophic activities; without access to sunlight, their growth is limited [13,14]. The most common identified algal genera include Chlorella, Stichococcus, Gloeocystis, Tetracystis, Cystomonas, Muriella, Scenedesmus, Klebsormidium, Apatococcus, and Trebouxia [15,16,17,18,19]. The removal of algae from building materials has been receiving increasing attention, with structural and technical methods of preventing algae growth and the use of various impregnation products being considered. However, commercial products currently in use are often not ecological because they contain various metals or organic pollutants. These compounds and elements are persistent, can accumulate in living organisms, and may be toxic to them. The goal of this study is therefore to find the most ecological ways to remove algae from building materials, ideally preventing their growth altogether [20,21,22,23,24,25].

One possibility is to use organic substances produced by plants to protect themselves against pathogens, such as bacteria, viruses, molds, fungi, nematodes, and beetle larvae [26,27,28,29]. One such group is essential oils. These substances and oils tend to be volatile, poorly soluble in water, and have an intense aroma. Since essential oils generally have an antimicrobial effect in small quantities, they may also be effective as algicidal agents. They are organic substances that degrade over time and therefore do not accumulate in the environment. They can be extracted directly from plants or produced synthetically. The biocidal effects of essential oils are already used across various industrial sectors for pests.

Essential oils are now used extensively, mainly in the food, perfume, and clothing industries. The most commonly used essential oils are derived from tropical or Mediterranean plants, such as tea tree, grapefruit, orange, lavender, and thyme. These oils contain various substances at different concentrations, which determine their properties and effectiveness [30,31]. From a chemical point of view, the most important groups of essential oil components are terpenoids, alcoholic compounds, acidic compounds, aldehydes, ketones, and aromatic phenols [32]. There are more than 100,000 published articles on the pesticidal/biocidal effects of essential oils in the Web of Science database. However, most focus is on fungi, bacteria, or molds in the context of building materials [16,26,30,33,34,35,36,37,38,39,40,41,42,43]. Very few studies primarily investigate the effects of essential oils on green algae [44,45,46,47,48,49,50].

In the first study, the authors investigated the efficacy of oregano essential oil and eugenol when applied directly in solution or encapsulated within silica nano-capsules against Chlorococcus sp. in a 100-day in vitro test. Both chemicals showed potential for use in protective biocidal coatings for stone artifacts [44]. In another study, Macchia et al. examined the impact of eucalyptus, basil, cloves, thyme, pine, and tea tree oil and their combinations on a natural biofilm containing algae. The most effective essential oils were tea tree, pine, and thyme oils in mixtures in their study [45]. Other authors investigated the effect of carvacrol and thymol on green algae growing on model ceramics, marble, and cement grit. They found that a mixture of these substances, together with an emulsifier (kaolin) and a gelling agent (laponite), was effective against the biofilm and destroyed it. The biofilm was then removed with the emulsion. The authors state that the disadvantage of this emulsion is its odor [46]. In another study, the authors focused on the use of essential oils (EOs) from Thymus vulgaris and Oreganum vulgare plants against bacteria, fungi, cyanobacteria, and green algae (Chlorella). Oregano oil was found to be more effective than thyme oil against algae [47]. Spada et al. [48] studied the effectiveness of carvacrol, eugenol, trans-cinnamon aldehyde, and thymol. They prepared various mixtures of essential oils, other chemicals, and carriers. The authors claimed that their mixtures enhanced biocidal action without damaging the stone substrate, enabling the use of low concentrations. In their final study, they isolated algal species from biofilms formed on deteriorating stone.

The new directions of the present study, compared with previously published papers, are as follows:

(1) Various essential oils have thus far been tested primarily in natural biofilms. This study aimed to compare the sensitivity of three model algae species and a laboratory-produced mixed artificial biofilm. The selected species are widespread algae commonly found on building stones or cultivated in bioreactors for the production of their metabolites. The use of artificially created biofilm (from abundant green species on building materials) aims to facilitate research into the use of biofilms from acquired sources, because it is very difficult to adequately cultivate biofilm removed from in situ in a laboratory environment—a selection always occurs, and it is not possible to cultivate all species of organisms that naturally occur in a natural biofilm. The results of the current study were then discussed and compared with data from the literature on natural biofilms.

(2) Thyme oil, thymol, carvacrol, and oregano oil are among the most studied oils in the construction industry. In this study, in addition to these substances, the algicidal effect of hops and caryophyllene was tested for the first time. Hop and caryophyllene were tested only as pesticidal substances in agriculture, not in building research.

(3) The algicidal effect of the substances was also discussed in terms of the chemical structure of the substances tested (thymol, carvacrol, trans-cinnamaldehyde, and caryophyllene), which are found in essential oils.

2. Materials and Methods

2.1. Chemicals and Reagents

The essential oils (cinnamon bark oil, thyme oil, and oregano oil) were purchased from the Ingredients Store (United Kingdom). Trans-cinnamaldehyde, carvacrol, thymol, and β-caryophyllene were purchased from Sigma-Aldrich Ltd. (Prague, Czech Republic).

Dimethyl sulfoxide (DMSO) (Sigma-Aldrich Ltd., Czech Republic) was used as a solvent at a ratio of 1:5. The dissolved essential substances were then added to the algal BBM medium. Deionized water was used as a solvent to prepare the BBM culture medium (CCALA—Czech Academy of Sciences, Třeboň, Czech Republic).

Reference substance for ecotoxicological tests with algae, potassium dichromate (K₂Cr₂O₇) and reference biocide terbutryne were purchased from Sigma-Aldrich Ltd. (Czech Republic).

2.2. Extraction of Hop Essential Oil

The hop oil was obtained by steam distillation from 2 kg of hop pellets, which were purchased from HopProducts CZ (Čepirohy, Czech Republic). The pellets were placed in a 65-liter essential oil extractor (Albrigi Luigi, Italy) together with 5 L of water. The pellets were separated from the water by a double-perforated bottom. The essential oil was collected from the separator and stored at 6 °C for further use after 2 h of distillation. The composition of the test essential oils is shown in

Table 1. Main compounds of the tested essential oils.

Essential Oil	Composition of Individual Essential Oils (%)
Cinnamon	70.7% trans-cinnamaldehyde, 8.8% Acetic acid, cinnamyl ester, 5.1% Eugenol
Thyme	43.4% p-Cymene, 42.4% Thymol
Oregano	65.3% Carvacrol, 17.8% p-Cymene
Hop	34.22% Humulene, 19.9% Humulene epoxide, 12.9% ß-Myrcene, 8.0% Caryophyllene oxide, 6.4% ß Caryophyllene

.

2.3. Chemical Analysis of the Studied Essential Oils

The composition of the essential oils was analyzed using an Agilent 7890A gas chromatograph coupled with a 5975C single quadrupole mass spectrometer (Agilent, Santa Clara, CA, USA). Separation was performed on a VF-5 MS column (30 m × 0.25 mm × 0.25 µm) with helium as the carrier gas (1 mL/min). Samples (1 µL) were injected in splitless mode at 250 °C. The oven temperature was programmed from 70 °C (2 min hold) to 230 °C at 5 °C/min, with a final 2 min hold. Ion source and quadrupole temperatures were set at 230 °C and 150 °C, respectively. Mass spectra were recorded in scan mode (m/z 35–550), and compounds were identified by comparison with reference standards, the NIST library, and Kovats retention indices.

Relative quantification was performed using the same GC system equipped with a flame ionization detector under identical conditions. Compound amounts were expressed as the ratio of individual peak area to the total peak area.

2.4. Organisms

Hematococcus pluvialis, Chlorella mirabilis, Stichococcus sp., and the BBM salt stock solution were purchased from CCALA (Czech Academy of Sciences, Třeboň, Czech Republic). The artificial biofilm consisted of the aforementioned species at a ratio of 1:1:1 (Figure 1, Figure 2 and Figure 3).

2.5. Bioassay

Stock solutions of all the tested species were cultured in a thermostat at a temperature of 22 ± 2 °C with a light intensity of 6000–8000 Lux. The control medium (Bold’s basal medium, BBM) with a pH of 7.0 ± 0.2 was prepared according to Bold.

The test methodology was adapted according to the number of substances tested and the number of model algae species. Therefore, the tests were performed in plastic microplates, with the density of the solution being measured as an indicator of algal cell concentration. As each model species has slightly different growth requirements, a compromise was chosen, and the tests were extended from the standard 72 h to 14 days to allow sufficient biomass to be produced by all the test species. The tests were carried out at gradually increasing temperatures (20 °C for the first two days and 22 °C for the remainder of the test, according to our preliminary practical experience—unpublished).

The test was carried out in plastic microplates, with a volume of 5 mL per well. Five replicates of the control medium and the individual oil concentrations were prepared. The experiment was performed at concentrations of 0 mg/L (control), 50 mg/L, 100 mg/L, and 200 mg/L. A subsequent experiment was performed with concentrations of 0, 3, 6, 12, and 25 mg/L of the most effective chemicals, based on the previous experiment. The appropriate amount of control medium and algal biomass (control) + individual test essential oil or oil substance, diluted in DMSO in a ratio of 1:5, were pipetted into each hole of the microplates. No negative effect of DMSO on algae was confirmed in our previous study [49]. The microplates were incubated in a thermostat under defined test conditions over a period of 14 days.

Algal growth was expressed as absorbance values. These values were measured using a YENWAY 6300 spectrophotometer at a wavelength of 750 nm [50].

Absorbances of the controls and concentrations were measured at the start (0 h) and end (336 h) of the experiments. The initial algal absorbance values were about 50 each time at the start of the experiments. The control medium and concentrations were incubated under light of 2000–4000 Lux at a temperature of 22 ± 2 °C. The measured absorbance values were used to calculate algal growth. The MIC values (the concentrations that caused 100% inhibition of growth) were determined after the experiments were completed.

2.6. Calculations

The absorbance values used in the inhibition calculations were estimated for each microplate (controls and tested concentrations) using the following equation:

A_final = (A_time336h − A_time0h)

(1)

MIC values corresponded to A_final = 0 ± 0.010.

2.7. Statistical Evaluation

The comparison of species sensitivity and the toxicity of EOs and substances at different concentrations was statistically evaluated using the nonparametric Dunn test (level of significance p > 0.005). The comparison of algicidal effects between the concrete EO and its paired substance was statistically evaluated by the nonparametric Mann–Whitney test (level of significance p > 0.005). The used statistical software was GraphPad Instat, version 3.0 (San Diego, CA, USA).

3. Result

3.1. Chemical Composition of Essential Oils

The essential oils were analyzed by GS-MS. Their chemical composition is shown in Table 1.

3.2. Algicidal Effects

Three species of green algae were used in the bioassays of the present study. Chlorella sp. is the dominant species, reproducing quickly and able to increase its biomass in various environments, including rivers, ponds, stones, and soils. It is therefore also used commercially for producing chlorophyll. Hematococcus pluvialis is another abundant species with large cells that is widespread in a variety of environments. Stichococcus sp. is cultivated for its fatty acid production for gas generation.

The reference chemical for performance of ecotoxicological tests with green algae—K₂Cr₂O₇ was tested on all model species and their mixture–biofilm, under the same conditions as is usual in the appropriate guideline [50]. The results indicate that the most sensitive species was Ch. mirabilis and the least sensitive H. pluvialis (Figure 4), but H. pluvialis was able to produce the highest biomass. The biofilm sensitivity was somewhere in the middle, thanks to the presence of less or more sensitive species in the mass, but its own growth rate was the least. The primary data are in Supplement No. S3.

In addition, the test with the standard commercial herbicide Terbutryne was conducted on the most sensitive species, Ch. mirabilis. The results are in Supplement No. S3.

The increase in biomass in the control group of the test with EOs and their components varied, but all species met the validity criteria of the algae test according to the standard at the end of the experiments [50]. The primary data—absorbance values (A) for each microplate and well—are presented in

Table 2. Primary data + mean values and standard deviations for algal tests with eight chemicals: A = cinnamon oil, B = thyme oil, C = oregano oil, D = hop oil, E = trans-cinnamon aldehyde, F = thymol, G = carvacrol, H = β-caryophyllene and the alga Hematococcus pluvialis.

Replicate	A	B	C	D	E	F	G	H
Control
1	0.261	0.154	0.242	0.193	0.261	0.228	0.430	0.239
2	0.288	0.177	0.200	0.185	0.340	0.339	0.470	0.323
3	0.268	0.182	0.194	0.195	0.252	0.274	0.521	0.311
4	0.265	0.171	0.166	0.186	0.214	0.256	0.682	0.341
5	0.253	0.205	0.203	0.223	0.281	0.234	0.657	0.338
mean	0.267	0.179	0.201	0.196	0.270	0.266	0.552	0.310
SD	0.013	0.019	0.027	0.016	0.046	0.045	0.112	0.042
50 mg/L
1	0.139	0.102	0.143	0.157	0.182	0.217	0.104	0.199
2	0.085	0.098	0.092	0.154	0.204	0.169	0.104	0.241
3	0.104	0.096	0.082	0.254	0.192	0.217	0.104	0.207
4	0.122	0.095	0.106	0.263	0.211	0.146	0.111	0.279
5	0.125	0.098	0.155	0.306	0.206	0.176	0.105	0.309
mean	0.116	0.098	0.116	0.227	0.199	0.185	0.106	0.247
SD	0.021	0.003	0.032	0.068	0.012	0.031	0.003	0.047
100 mg/L
1	0.076	0.098	0.083	0.215	0.245	0.224	0.114	0.204
2	0.144	0.097	0.080	0.213	0.135	0.188	0.107	0.238
3	0.085	0.086	0.083	0.159	0.151	0.207	0.103	0.331
4	0.139	0.099	0.090	0.162	0.282	0.148	0.104	0.337
5	0.094	0.098	0.085	0.155	0.282	0.161	0.108	0.344
mean	0.108	0.096	0.084	0.181	0.203	0.186	0.107	0.291
SD	0.032	0.005	0.004	0.030	0.072	0.032	0.004	0.065
200 mg/L
1	0.117	0.090	0.085	0.159	0.165	0.251	0.114	0.198
2	0.084	0.093	0.086	0.144	0.147	0.197	0.107	0.287
3	0.083	0.096	0.085	0.141	0.306	0.188	0.101	0.295
4	0.084	0.095	0.090	0.137	0.199	0.167	0.104	0.313
5	0.085	0.097	0.085	0.135	0.162	0.165	0.108	0.321
mean	0.091	0.094	0.086	0.143	0.196	0.194	0.107	0.283
SD	0.015	0.003	0.002	0.010	0.065	0.035	0.005	0.050

,

Table 3. Primary data + mean values and standard deviations for algal tests with eight chemicals: A = cinnamon oil, B = thyme oil, C = oregano oil, D = hop oil, E = trans-cinnamon aldehyde, F = thymol, G = carvacrol, H = β-caryophyllene and the alga Chlorella mirabilis.

Replicate	A	B	C	D	E	F	G	H
Control
1	0.387	0.503	0.554	0.265	0.298	0.193	0.160	0.381
2	0.474	0.621	0.583	0.537	0.483	0.182	0.174	0.374
3	0.545	0.783	0.523	0.377	0.380	0.243	0.176	0.359
4	0.498	0.651	0.418	0.233	0.357	0.255	0.167	0.338
5	0.445	0.591	0.458	0.216	0.288	0.167	0.155	0.340
mean	0.470	0.630	0.507	0.326	0.362	0.208	0.166	0.358
SD	0.059	0.102	0.068	0.134	0.078	0.039	0.009	0.019
50 mg/L
1	0.137	0.185	0.091	0.332	0.178	0.174	under limit of detection	0.344
2	0.165	0.251	0.105	0.220	0.125	0.111		0.361
3	0.114	0.219	0.086	0.243	0.138	0.129		0.340
4	0.106	0.216	0.083	0.253	0.148	0.125		0.432
5	0.145	0.204	0.115	0.407	0.142	0.145		0.491
mean	0.133	0.215	0.096	0.291	0.146	0.137		0.394
SD	0.024	0.024	0.014	0.077	0.020	0.024		0.066
100 mg/L
1	0.117	0.119	0.079	0.260	0.112	0.167	values under limit of detection	0.291
2	0.113	0.109	0.082	0.267	0.185	0.088		0.274
3	0.112	0.132	0.085	0.178	0.120	0.010		0.337
4	0.114	0.132	0.108	0.192	0.116	0.087		0.399
5	0.139	0.121	0.103	0.138	0.142	0.160		0.401
mean	0.119	0.123	0.091	0.207	0.135	0.120		0.340
SD	0.011	0.010	0.013	0.055	0.030	0.040		0.059
200 mg/L
1	0.136	0.086	0.079	0.332	0.121	1.283	values under limit of detection	0.177
2	0.109	0.085	0.084	0.246	0.120	1.405		0.266
3	0.109	0.082	0.078	0.258	0.115	0.994		0.266
4	0.163	0.083	0.072	0.233	0.117	1.248		0.302
5	0.152	0.087	0.076	0.373	0.117	1.271		0.286
mean	0.134	0.085	0.079	0.288	0.118	1.240		0.259
SD	0.025	0.002	0.003	0.061	0.003	0.151		0.049

,

Table 4. Primary data + mean values and standard deviations for algal tests with eight chemicals: A = cinnamon oil, B = thyme oil, C = oregano oil, D = hop oil, E = trans-cinnamon aldehyde, F = thymol, G = carvacrol, H = β-caryophyllene and the alga Stichococcus sp.

Replicate	A	B	C	D	E	F	G	H
Control
1	0.331	0.310	0.276	0.391	0.430	0.772	0.794	0.344
2	0.312	0.314	0.363	0.357	0.347	0.730	1.053	0.375
3	0.324	0.341	0.361	0.400	0.326	0.640	0.959	0.416
4	0.344	0.334	0.319	0.363	0.234	0.647	1.092	0.337
5	0.303	0.308	0.313	0.400	0.260	0.741	1.150	0.340
mean	0.323	0.321	0.326	0.378	0.319	0.706	1.010	0.362
SD	0.016	0.015	0.037	0.021	0.077	0.059	0.139	0.300
50 mg/L
1	0.132	values under limit of detection	0.100	0.233	0.094	1.042	0.130	0.386
2	0.083		0.088	0.186	0.089	1.088	0.140	0.358
3	0.077		0.094	0.155	0.085	0.709	0.143	0.363
4	0.073		0.087	0.148	0.085	0.968	0.133	0.307
5	0.078		0.092	0.155	0.083	1.185	0.135	0.310
mean	0.089		0.092	0.181	0.087	0.998	0.136	0.345
SD	0.025		0.005	0.039	0.004	0.180	0.005	0.035
100 mg/L
1	0.097	values under limit of detection	0.089	0.260	0.090	1.516	0.134	0.372
2	0.078		0.089	0.169	0.089	0.791	0.124	0.403
3	0.117		0.075	0.181	0.089	1.203	0.106	0.324
4	0.160		0.089	0.127	0.091	1.057	0.131	0.296
5	0.095		0.090	0.181	0.088	1.536	0.148	0.290
mean	0.109		0.086	0.184	0.089	1.221	0.129	0.337
SD	0.031		0.006	0.056	0.001	0.316	0.015	0.049
200 mg/L
1	0.076	values under limit of detection	0.081	0.172	0.093	1.283	0.134	0.377
2	0.084		0.086	0.130	0.090	1.405	0.145	0.386
3	0.134		0.082	0.121	0.090	0.994	0.133	0.388
4	0.121		0.085	0.120	0.084	1.248	0.138	0.365
5	0.112		0.077	0.172	0.090	1.271	0.129	0.301
mean	0.107		0.082	0.136	0.089	1.240	0.136	0.363
SD	0.025		0.004	0.025	0.003	0.151	0.006	0.039

and

Table 5. Primary data + mean values and standard deviations for algal tests with eight chemicals: A = cinnamon oil, B = thyme oil, C = oregano oil, D = hop oil, E = trans-cinnamon aldehyde, F = thymol, G = carvacrol, H = β-caryophyllene, and a biofilm consisting of 3 species (H. pluvialis, Ch. m., S. sp.). Absorbance values at the time 0 were up to 50.

Replicate	A	B	C	D	E	F	G	H
Control
1	0.331	0.310	0.276	0.391	0.430	0.772	0.794	0.344
2	0.312	0.314	0.363	0.357	0.347	0.730	1.053	0.375
3	0.324	0.341	0.361	0.400	0.326	0.640	0.959	0.416
4	0.344	0.334	0.319	0.363	0.234	0.647	1.092	0.337
5	0.303	0.308	0.313	0.400	0.260	0.741	1.150	0.340
mean	0.323	0.321	0.326	0.378	0.319	0.706	1.010	0.362
SD	0.016	0.015	0.037	0.021	0.077	0.059	0.139	0.304
50 mg/L
1	0.132	values under limit of detection	0.100	0.233	0.094	1.042	0.130	0.386
2	0.083		0.088	0.186	0.089	1.088	0.140	0.358
3	0.077		0.094	0.155	0.085	0.709	0.143	0.363
4	0.073		0.087	0.148	0.085	0.968	0.133	0.307
5	0.078		0.092	0.155	0.083	1.185	0.135	0.310
mean	0.089		0.092	0.181	0.087	0.998	0.136	0.345
SD	0.025		0.005	0.039	0.004	0.180	0.005	0.035
100 mg/L
1	0.097	values under limit of detection	0.089	0.260	0.090	1.516	0.134	0.372
2	0.078		0.089	0.169	0.089	0.791	0.124	0.403
3	0.117		0.075	0.181	0.089	1.203	0.106	0.324
4	0.160		0.089	0.127	0.091	1.057	0.131	0.296
5	0.095		0.090	0.181	0.088	1.536	0.148	0.290
mean	0.109		0.086	0.184	0.089	1.221	0.129	0.337
SD	0.031		0.006	0.056	0.001	0.316	0.015	0.049
200 mg/L
1	0.076	values under limit of detection	0.081	0.172	0.093	1.283	0.134	0.377
2	0.084		0.086	0.130	0.090	1.405	0.145	0.386
3	0.134		0.082	0.121	0.090	0.994	0.133	0.388
4	0.121		0.085	0.120	0.084	1.248	0.138	0.365
5	0.112		0.077	0.172	0.090	1.271	0.129	0.301
mean	107		0.082	0.136	0.089	1.240	0.136	0.363
SD	0.025		0.004	0.025	0.003	0.151	0.006	0.039

for all test chemicals and organisms. Absorbance values were also measured for solutions containing substances/oils in the absence of algae, with values not exceeding 0.020. The results are included in Supplement No. S1.

Based on the initial data, it was found that hop oil, caryophyllene and thymol did not exhibit any significant toxic effects on organisms at any of the tested concentrations. Cinnamon oil, trans-cinnamon aldehyde, and oregano oil exhibited variable toxicity to certain species and biofilms, though MIC values were not reached. These substances were therefore not tested further. Thyme oil and carvacrol were only tested at concentrations of 25, 12, 6, and 3 mg/L, as these chemicals caused MIC of 50 or 100 mg/L for some of the species and biofilms (Table 3, Table 4 and Table 5).

The results of the second experiment indicate that thyme oil is not toxic within the tested concentration range (see

Table 6. Absorbance values and their standard deviations for tests involving thyme oil, Stichococcus sp., and the biofilm.

Thyme Oil
Species	S. sp.
Concentration	control	3 mg/L	6 mg/L	12 mg/L	25 mg/L
1	1.970	1.834	1.829	1.825	1.486
2	1.945	1.415	1.438	1.394	1.992
3	1.932	1.297	1.028	1.510	1.799
4	1.908	1.312	0.802	1.485	1.986
5	1.962	1.860	1.632	1.699	1.978
mean	1.939	1.465	1.274	1.554	1.816
SD	0.026	0.252	0.454	0.188	0.237
Species	Biofilm
Concentration	control	3 mg/L	6 mg/L	12 mg/L	25 mg/L
1	0.639	0.662	0.694	0.616	0.616
2	0.458	0.510	0.570	0.550	0.474
3	0.443	0.479	0.499	0.439	0.497
4	0.419	0.471	0.512	0.425	0.497
5	0.476	0.180	0.444	0.267	0.474
mean	0.487	0.460	0.544	0.459	0.529
SD	0.078	0.156	0.085	0.120	0.062

). Initially, the carvacrol solutions (3, 6, 12, and 25 mg/L) were colorless. However, during the test, they began to turn pink and were mostly red by the end. This indicates the presence of carotenoids in these holes (see Supplement No. S2). No algal cells were observed under a light microscope in the plates, and thus, the resulting solutions were not evaluated. Additionally, carvacrol exhibited a strong aroma even at the lowest tested concentration of 3 mg/L.

Table 7. The MIC concentration values (mg/L) of oil chemicals for each algal species and biofilm are shown below. Notes: A = cinnamon oil, B = thyme oil, C = oregano oil, D = hop oil, E = trans-cinnamon aldehyde, F = thymol, G = carvacrol, H = β-caryophyllene.

	A	B	C	D	E	F	G	H
Species
Ch. m.	>200	>200	200	>200	>200	>200	>200	50
H. p.	>200	>200	>200	>200	>200	>200	>200	>200
S. sp.	>200	50	>200	>200	>200	>200	>200	>200
Biofilm	>200	50	>200	>200	>200	>200	>200	50

shows the MIC (maximum inhibitory concentration) values for all the chemicals and species tested. It is evident that the sensitivity of the algal species varied to some extent. The lowest effective concentrations (LOEC) were statistically verified (

Table 8. The lowest effective tested concentration (LOEC) values (mg/L) of oil chemicals for individual algal species and biofilms are shown below. These values were statistically verified using Dunnett’s test at α level of 0.05. Statistical analyses were performed with GraphPad InStat software, version 3, using Dunn’s Multiple Comparisons Test (GraphPad Software, San Diego, CA, USA). The calculations are available in the Supplement (Excel S1—a list named LOEC).

Species	A	B	C	D	E	F	G	H
Ch. m.	100	100	100	200	>200	100	50	3 *
H. p.	200	200	200	200	100	>200	50	3 *
S. sp.	50	50	200	>200	50	100	100	3 *
Biofilm	50	50	200	200	50	100	100	3 *

*—The value was selected as the LOEC values, because the algae were not able to live in the solution.

) and revealed earlier, indicating more variable species sensitivity than the MIC values.

3.3. Sensitivity of Algal Species

Graphs 5A–5H in Figure 5 show the sensitivity of individual species to the tested compounds/essential oils. It is evident that sensitivity differs between species. H. pluvialis appears to be the least sensitive species, while the biofilm, Ch. m., and S. sp. were more sensitive than H. pluvialis. The most toxic chemicals at lower concentrations were thyme oil and carvacrol, as shown in Table 8.

3.4. Effects on the Base of Chemical Composition and Structure

The statistical difference between pairs cinnamon oil-trans–cinnamon aldehyde, thyme oil–thymol, oregano oil–carvacrol, and hop oil–caryophyllene was verified using the Mann–Whitney Test (see

Table 9. U-values and p-values of the corresponding oil–compound pairs and the level of difference expressed by these values. The comparisons were performed using the Mann–Whitney test (InStat, GraphPad software, Vesion 3).

Pair	U’	Difference	p-Value
cinnamon oil x trans-cinnamon aldehyde	90.00	no	0.3120
thyme oil x thymol	134.00	extremely significant	0.0001
oregano oil x carvacrol	136.50	extremely significant	0.0002
hop oil x caryophyllene	117.50	very significant	0.0094

and, for more details, also Supplement S1, Excel list named “oils contra compounds”). The data indicated no significant difference between cinnamon oil and trans-cinnamon aldehyde. The other pairs were either significantly (hop oil and caryophyllene) or extremely significantly different (thyme oil and thymol, oregano oil and carvacrol).

4. Discussion

Comparison of their composition with other essential oils from previously published studies is not very easy, because individual oils always less or more differ slightly in their composition and percentage of individual substances, depending on the growing conditions, area of location (mainly country) and the method of extraction of oils from plants. Nevertheless, some correlations were confirmed. Some authors described similar levels of thymol in their thyme oil—47.6% [51,52] but a lower amount of p-cymene (about 8%). A total of 30 constituents of thyme oil were identified: thymol (39.44%), P-cymene (23.6%), and γ-terpinene (12.51%) were the main components comprising 79.91% of the thyme oil in a study of Abozid and Asker [53]. Dong et al. [54] also detected thymol as a main compound in the tested thyme oil. The main component of the oregano oils was carvacrol (76.64–85.70%) in a study of Walasek-Janusz et al. [55]. These authors wrote that mainly the Polish essential oils consisted of the highest levels of carvacrol in the oregano oils. The composition of our analyzed hop essential oil did not significantly change from the others [56,57]. The other authors analyzed the composition of cinnamon bark oil [58]. Their results show that (E)-cinnamaldehyde (71.50%), linalool (7.00%), β-caryophyllene (6.40%), eucalyptol (5.40%), and eugenol (4.60%) in their EO are similar to our results.

The sensitivity of species to the reference substance potassium dichromate was on the same level as for standard species Desmodesmus subspicatus. The species used were less sensitive in comparison to the species Selenastrum capricornutum. Chlorella mirabilis as the most sensitive species in the present study was selected as the species for test with model herbicide Terbutryne. Ch. mirabilis was more sensitive to herbicide than Pseudokirchneriela subcapitata but similarly sensitive as D. subspicatus (see Supplement No. S3).

The results indicate that carvacrol (5-isopropyl-2-methylphenol), which belongs to the monoterpenes group (see

Table 10. Physical and chemical properties of carvacrol, thymol, trans-cinnamaldehyde, and caryophyllene. These values were obtained from https://pubchem.ncbi.nlm.nih.gov/.

Substance	Carvacrol	Thymol	Cin. Aldehyde	Caryophyllene
Chemical structure
Systematic name	5-isopropyl-2-methylfenol	5-methyl-2-(propan-2-yl)fenol	(2E)-3-fenylprop-2-enal	(1R,4E,9S)-4,11,11-Trimethyl-8-methylidenebicyclo[7.2.0]undec-4-ene
Molecular weight	150.22 g/mol	150.22 g/mol	132.16 g/mol	204.36 g/mol
log Kow	3.10	3.30	1.90	6.23
Solubility in water	1.25 g/L	0.9 g/L	1.42 g/L	˂1 g/L
Electrophilicity	Donor of el.	Donor of el.	Acceptor of el.	Donor of el.
Refractive index	1.523	1.522	1.619–1.627	1.492–1.502
Dipole moment	1.4381 D	1.893 D	4.762–5.1 D	0
Energy gap	4.8–5.0 eV	5.5 eV	4.371 eV	0

), was the most effective compound against all the tested species. Monoterpenes are considered secondary plant metabolites and function as attractants, morpho-regulators, or protective substances [59]. Carvacrol can react with hydroxyl radicals, leading to oxidative stress [60]. It has been found to reduce the expression of genes that encode antioxidant enzymes, influencing the production and activity of antistress enzymes [61]. Other research has confirmed that carvacrol causes cell death by decreasing ergosterol, which leads to membrane destruction [62]. In another study, EO mixtures exhibited higher biocide activity than individual EOs due to their synergistic effects against biofilms containing Chlorella on stone surfaces [63]. In the present study, the effect of carvacrol was observed as color changes, with biofilm and Stichococcus cultures turning red (Supplement S2). It indicates the production of protective pigments, such as carotenoids, which are indicators of oxidative stress. In addition, a comparison experiment confirming carotenoid production by the used model species was performed, and the results are presented in Supplement S1.

Conversely, strong similarities were found between cinnamon oil and trans-cinnamon aldehyde, as the tested cinnamon oil had a very high concentration of trans-cinnamon aldehyde in the present study (see Figure 3, Table 1 and Table 9). The tested hop oil contained only around 8% caryophyllene, but its slightly higher toxicity appears to be caused by other components, likely humulene (Table 1). The low toxicity of oregano oil compared to carvacrol was surprising, but this may be due to the presence of 17.8% p-Cymene in the tested oil and its interactions with the other components. Similarly, some discrepancies were observed between toxic thyme oil and non-toxic thymol. It can be assumed that the presence of 43.4% p-Cymene affects the overall toxicity of the thyme oil tested.

The robust QSAR methodology could not be used in the present study because the tested essential oils contained many different chemicals, and the individual test substances were quite structurally dissimilar (e.g., monoterpenes such as thymol and carvacrol, trans-cinnamon aldehyde, and the bicyclic sesquiterpene caryophyllene). The study only discusses a simple dual structure–activity relationship (SAR). This method links a single aspect of the chemical structure to the observed biological activity. Some authors have described how the biological activity of a compound is directly proportional to its hydrophobicity, with the cytoplasmic membrane being the primary site of toxicity [63,64], while others have suggested that molecular size, hydrophobicity, polar surface area (PSA), log K_ow values, and hydrogen bond donor capacity can effectively describe chemical–biological interactions [65]. In the present study, no clear property–activity relationship was observed at any significant level, likely due to the small number of chemicals tested. Only lower lipophilicity (log K_ow < 3.1) and higher water solubility (>1 g/L) were partial factors in the higher toxicity of carvacrol and trans-cinnamon aldehyde, as opposed to thymol and caryophyllene (Table 10). Conversely, hop oil and caryophyllene were not toxic, likely because the large size of caryophyllene limited its bioavailability to algae. It was unable to penetrate the algal cell walls and membranes and destroy them, consistent with previous findings that membrane accessibility is limited to compounds with log p values below 5 and molecular weights ≤ 500 [65].

Interestingly, the anti-algal activity of carvacrol and thymol differs, even though these compounds are isomers with the same molecular weight and other properties (Table 10). The main differences are likely to be found in the dipole moment or energy gap, which indicates the higher toxicity of carvacrol. Another possible factor is the bond dissociation enthalpy of the phenolic O–H bond (ΔH) in carvacrol than in thymol [66]. Carvacrol’s enthalpy is about 85.4 kcal/mol, whereas thymol’s is lower at approximately 80.3 kcal/mol. The effect of dissociation enthalpy is explained in detail in [66], which states that free hydroxyl groups lead to proton exchange, disturbing the membrane gradient and ultimately resulting in cell rupture. It also states that the relative position of the hydroxyl group on the phenolic ring influences the antimicrobial efficacy of these molecules. Another study confirmed that carvacrol is more effective than its isomer, thymol, and that their combination did produce a synergistic effect at the tested concentrations [67]. Furthermore, the reaction of other organisms, such as earthworms, Vibrio (sea bacteria), and Daphnia magna (crustaceans), was again more pronounced for carvacrol than for thymol [68].

The different sensitivities of individual algal species are mainly due to their size or ecology. As can be seen in Figure 2, H. pluvialis has significantly larger cells than Stichococcus sp. or Chlorella mirabilis, which could explain why it is less accessible to substances or essential oils. H. pluvialis can produce carotenoids under stressful conditions, including the red-colored astaxanthin, which is considered the most powerful natural antioxidant. This protects cell membranes from damage caused by UV radiation and chemical oxidants and is often commercially cultivated under stress conditions to produce as much of this pigment as possible [69]. Chlorella sp. generally contains higher levels of chlorophyll than other green algae. This pigment plays a key role in photosynthesis, and its antioxidant properties have been demonstrated in numerous studies (e.g., [69,70,71]. Conversely, some species of the genus Stichococcus are cultivated for their fatty acid production. Stichococcus sp. is likely unable to produce such a large quantity of protective metabolites, and its small molecules may be more sensitive to essential oils [72]. The measured concentrations of total carotenoids were analyzed spectrometrically for all three model species in the present study. The results suggest that carotenoid production is higher during the resting phase of H. pluvialis culturing than during the culturing of C. mirabilis and S. sp., indicating that carotenoids may play a more important role in algal tolerance of essential oils. (Supplement S1).

One Italian study found that cinnamon, oregano, and thyme essential oils (EOs) at a concentration of 5% (v/v) were the most effective against microorganisms, including green algae from the genus Chlorella isolated from biofilms in the Colosseum [48]. In another Italian study, the authors measured lower algae biomass on marble using colorimetry after applying a gel containing 0.25% thyme oil. Only a single treatment was needed to completely remove these microorganisms. The biomass consisted of various green algae species (Chlorella sp.) [73]. Candela et al. [53] applied emulsion systems containing an essential oil/water ratio of 1:3, stabilized with 4% kaolinite and Laponite^®, against natural biofilms. This formulation eliminated algae from treated surfaces without leaving residues, and the effect persisted for up to four months, according to the authors. Finally, Kobetičová et al. [57] tested carvacrol, thyme oil, cinnamon oil, eugenol, and thymol on a natural biofilm isolated from lime–cement plaster. The results were consistent with the findings of the present study.

Our results are consistent with previous studies that have investigated the effectiveness of essential oils or their components on natural algal biofilms. As expected, the artificial biofilm in the present study exhibited sensitivity to the tested chemicals similar to that of the three individual species. Therefore, artificial biofilms can be used as a model for natural algal biofilms in future studies, since essential oils and their components generally have an anti-algal effect on many species of green algae that live on building facades, bricks or concrete.

5. Conclusions

In the present study, carvacrol (MIC = 3 mg/L) and thyme oil (50 mg/L) were found to be the most effective substances in enhancing the algacidal effect. Hop oil and caryophyllene were not toxic to algae at concentrations of up to 200 mg/L. The chemical structures of the test chemicals indicate that molecular size, log Kow, water solubility, and dipole moment influence their potential toxicity to algae. Hematococcus pluvialis was less sensitive than Ch. mirabilis and S. sp., likely due to its production of protective pigments. The results indicate that the artificial biofilm compost of the three green algal species (H. pluvialis, Ch. mirabilies, S. sp.) exhibited similar sensitivity to algal species isolated from natural conditions (from previous studies or the authors’ own work). This suggests that suitable artificial biofilms can replace natural biofilms, which are often difficult to cultivate in a laboratory setting. Limitations of the present study: the number of substances tested and compared was relatively small; however, future research could expand the range of essential substances for QSAR analysis. The tested EOs or their components were evaluated only on green algae, whereas natural biofilms consist of diverse microorganisms. Future research: Carvacrol was the most effective compound, but it produces an intense aroma and colors solutions. Therefore, future studies should focus more intensively on its fate in the environment and on building material surfaces. Its ecotoxicity will be tested, and its properties in coating systems will be studied in more detail.