Effects of Hypoxia on the Distribution of Calanoid Copepod Eggs in the Seabed Sediments of the Eutrophic Masan Bay, Korea

: In this study, we investigate the distribution of calanoid copepod eggs in the sediments of Masan Bay (South Sea, Korea), in which hypoxic conditions occur every summer. In August (2011), hypoxia was observed at all stations, while normoxia was observed in April (2012). The pH and hydrogen sulphide concentration at the sediment-water interface in the inner bay during August were 7.7 and >20 mol L − 1 , respectively, and there was no signiﬁcant difference between the stations in April. The abundance of eggs in the sediment ranged from 0.69 to 1.49 × 10 6 eggs m − 2 in August, and from 0.59 to 1.08 × 10 6 eggs m − 2 in April. Notably, the proportion of abnormal eggs was high (a maximum of 77.1%) in August, and a uniform distribution of normal eggs (>80%) was observed in April. A generation of abnormal eggs hatched with deformed nauplii in Masan Bay during the summer, and a failure to hatch was likely due to the high H 2 S concentration and low pH caused by hypoxia. In this paper, we discuss the effects of hypoxia and seabed environments on the hatching success of calanoid copepod eggs.


Introduction
Eutrophication is a problem that persists in coastal waters worldwide, including those in Korea, particularly Masan Bay [1].Several coastal ecosystems are directly and indirectly affected by bottom hypoxia (<2 mL L −1 dissolved oxygen (DO)) caused by increased anthropogenic eutrophication [2].In addition, oxygen-deficient sediments are frequently associated with the formation of hydrogen sulphide (H 2 S), which is toxic to most benthic organisms [3].In aqueous solutions, the equilibrium concentration of different sulphide species (i.e., H 2 S, HS − , and S 2− ) varies with the pH.The pH of natural seawater ranges from 7.9 to 8.3, and HS − is dominant.In pore water, where the pH can be as low as 6.0-6.5, the dominant species is H 2 S [4,5].The toxicity of sulphide to marine benthic organisms is controlled by pH, and it increases with a decrease in pH [4].
Most marine calanoid copepods spawn their eggs freely in the water column, which sink because their density is higher than that of the surrounding seawater [6].The spawned eggs can be subitaneous eggs that normally hatch within a few days without a delay, or diapause eggs that enter an obligatory refractory phase [7].Some calanoid species produce both egg types, while others produce only one type.Diapause egg hatching is important for the annual perpetuation of species that disappear entirely from the water column for certain periods in a year, while subitaneous egg hatching is important for the continuation of the populations within each active season [8,9].The highest number of calanoid copepod eggs (i.e., 10 6 eggs m −2 ) has been found in the bottom sediment of shallow waters (<20 m) [10].
Water 2021, 13, 3116 2 of 11 The accumulation of such large numbers of eggs forms an "egg bank," which plays an important role as a potential source of nauplii for recruitment [11][12][13].In shallow waters, copepod eggs often sink and can spend hours to years on the seabed before hatching [14].Therefore, the sinking eggs can be exposed to stressful conditions, such as hypoxia, anoxia, and sulphide stress.When copepod eggs are exposed to hypoxia or anoxia for a long time, the population dynamics of copepod species are adversely affected [8,[15][16][17].In addition, Invidia et al. [18] reported that these adverse effects can be stronger under a combination of low oxygen concentration and sulphide.
The Masan Bay is a semi-enclosed inner bay, located on the southeastern coast of Korea, and covers an area of 33.8 km 2 with an average depth of 15 m (ranging from 5 to 25 m) [1].It is surrounded by densely populated cities (such as Masan, Changwon, and Jinhae) with over one million inhabitants and several industrial complexes.Over the past few decades, domestic sewage and industrial effluents have continued to flow into the bay.As a result, the water quality of Masan Bay has severely deteriorated, and vast algal blooms occur annually [19,20].In addition, the occurrence of hypoxia or anoxia in the near-bottom sediment during summer (June-September) has increased annually [21].Masan Bay is a target area for intense research because it is the most eutrophic embayment in Korea.Most studies have focused on phytoplankton, fish, and benthos [21][22][23][24][25], while studies on copepod eggs have been limited to date.As hypoxia in the inner part of Masan Bay lasts for five months (June-September) [21,26], the settled eggs are likely affected by deteriorating conditions for a long period of time.
The purpose of this study is to investigate the environmental factors of the seabed and the copepod egg abundance to evaluate the effect of hypoxia on the copepod egg hatching rate in Masan Bay, wherein hypoxia is widespread during the summer.In addition, the effect of hypoxia was indirectly evaluated by identifying the distribution characteristics of normal and abnormal eggs.

Materials and Methods
The environmental factors of the seabed, copepod egg abundance, and egg shape normality were investigated at four sites in Masan Bay, from August 2011 to April 2012 (Figure 1).The physical factors (water temperature, salinity, and DO concentration), pH, and total H 2 S concentration in the sediment were investigated as the seabed environmental factors.The water temperature, salinity, and DO concentration of the seabed were measured using a water quality measurement system (Model 6600; Xylem Inc., Yellow Springs, OH, USA), which can record data by approaching the seabed as close as possible.The measurement of the DO concentration with the sensor involved a photocatalytic method, which was used after confirming the high correlation with the existing Winkler method.Surface sediment samples, to measure the pH and H 2 S concentration of the seabed, were collected using a sediment corer (area: 0.003 m 2 ) designed to prevent sediment disturbance.The sediment samples were immediately sealed after collection to prevent contact with air, and then transferred to the laboratory while maintaining the in situ water temperature in order to complete the measurement within 3 h.The vertical profile of pH and H 2 S in the sediment was measured using a microelectrode (Unisense, pH 500 and H 2 S 500, Aarhus, Denmark).After stabilisation and calibration, the microelectrode was mounted on a micromanipulator (Unisense, LS18, Aarhus, Denmark) with a vertical movement resolution of 10 mm, and directly inserted into the sediment by 100 mm to measure the pH and H 2 S concentration.The currents detected in the pH and H 2 S microelectrodes were subjected to a high sensitivity picoammeter (Unisense, PA2000, Aarhus, Denmark) and a digital converter (Unisense, pH/mV meter, Aarhus, Denmark), respectively, and the final concentration was calculated with a calibration curve.The method suggested by Cline [27] was used to prepare a standard solution of H 2 S and a calibration curve according to the current.
suggested by Cline [27] was used to prepare a standard solution of H2S and a calibration curve according to the current.The abundance of copepod eggs in the seabed was measured using a sediment corer.The sediment obtained by cutting the upper 0.5-1.0cm of the core was filtered through a 45 µ m mesh, and the remnants on the mesh were recovered, fixed in a formalin solution, and finally concentrated in a 50 mL tube in the laboratory.After collecting 1 mL of the concentrated sample in the Bogorov counting chamber and diluting it with filtered seawater, the copepod eggs were counted three times under a dissecting microscope (Olympus, SZX7, Tokyo, Japan), and the final abundance was converted to eggs per unit area (eggs m −2 ).The normality of the eggs was evaluated while simultaneously determining the egg abundance.In this study, the normal and abnormal eggs were distinguished based on the cracks in the eggshells and were measured by referring to the results of Blades-Eckelbarger and Marcus [28].The eggs that appeared to be physically damaged due to The abundance of copepod eggs in the seabed was measured using a sediment corer.The sediment obtained by cutting the upper 0.5-1.0cm of the core was filtered through a 45 µm mesh, and the remnants on the mesh were recovered, fixed in a formalin solution, and finally concentrated in a 50 mL tube in the laboratory.After collecting 1 mL of the concentrated sample in the Bogorov counting chamber and diluting it with filtered seawater, the copepod eggs were counted three times under a dissecting microscope (Olympus, SZX7, Tokyo, Japan), and the final abundance was converted to eggs per unit area (eggs m −2 ).The normality of the eggs was evaluated while simultaneously determining the egg abundance.In this study, the normal and abnormal eggs were distinguished based on the cracks in the eggshells and were measured by referring to the results of Blades-Eckelbarger and Marcus [28].The eggs that appeared to be physically damaged due to cracked shells or missing egg contents were considered abnormal [29,30] (Figure 2).Calanoid eggs were identified using morphological characteristics based on Kasahara et al. [31].

Environmental Variation
The water temperature variation was <2 °C in the bottom layer during August and <1 °C in April (Figure 3a).In addition, the salinity varied narrowly by <1.5 at the stations, during both months (Figure 3b).The salinity varied from 30.5 to 31.4 in August and from 33.0 to 33.6 in April, and was particularly low (<30.6) in the inner bay (Station1, 2) during August.The DO concentration formed a hypoxic water mass of <1 mL L −1 at all sites in August near the bottom (Figure 3c).In contrast, the DO concentration in April was within the normal range of 3.7-5.3mL L −1 .The DO concentration increased towards the outer bay during both months.

Sediment pH and H2S Concentration
The vertical sediment pH profiles are shown in

Environmental Variation
The water temperature variation was <2 • C in the bottom layer during August and <1 • C in April (Figure 3a).In addition, the salinity varied narrowly by <1.5 at the stations, during both months (Figure 3b).The salinity varied from 30.5 to 31.4 in August and from 33.0 to 33.6 in April, and was particularly low (<30.6) in the inner bay (Station 1, 2) during August.The DO concentration formed a hypoxic water mass of <1 mL L −1 at all sites in August near the bottom (Figure 3c).In contrast, the DO concentration in April was within the normal range of 3.7-5.3mL L −1 .The DO concentration increased towards the outer bay during both months.

Environmental Variation
The water temperature variation was <2 °C in the bottom layer during August and <1 °C in April (Figure 3a).In addition, the salinity varied narrowly by <1.5 at the stations, during both months (Figure 3b).The salinity varied from 30.5 to 31.4 in August and from 33.0 to 33.6 in April, and was particularly low (<30.6) in the inner bay (Station1, 2) during August.The DO concentration formed a hypoxic water mass of <1 mL L −1 at all sites in August near the bottom (Figure 3c).In contrast, the DO concentration in April was within the normal range of 3.7-5.3mL L −1 .The DO concentration increased towards the outer bay during both months.

Sediment pH and H2S Concentration
The vertical sediment pH profiles are shown in

Sediment pH and H 2 S Concentration
The vertical sediment pH profiles are shown in The total H2S concentration at the sediment-water interface was higher in August (0.34-59.66 μM) than that in April (0.06-8.16 μM) (Figure 4c,d).During the sampling period, the concentrations in the inner bays (Station 1 and 2) exceeded those at the other stations.The total H2S concentration sharply increased in the sediment-water interface towards a depth of 3 mm during August (Station 1, 2) and April (Station 1), and it was >100 μM at a depth of 1-6 mm.The total H 2 S concentration at the sediment-water interface was higher in August (0.34-59.66 µM) than that in April (0.06-8.16 µM) (Figure 4c,d).During the sampling period, the concentrations in the inner bays (Station 1 and 2) exceeded those at the other stations.The total H 2 S concentration sharply increased in the sediment-water interface towards a depth of 3 mm during August (Station 1, 2) and April (Station 1), and it was >100 µM at a depth of 1-6 mm.

Egg Abundance and Ratio According to Morphology (Normal and Abnormal)
The abundance of calanoid eggs ranged from 0.69 to 1.49 × 10 6 eggs m −2 during August and 0.59 to 1.08 × 10 6 eggs m −2 during April (Figure 5).The egg abundance was >1.00 × 10 6 eggs m −2 in August (Station 2, 3) and April (Station 1), and tended to increase towards the inner side of the bay during April.
Water 2021, 13, x FOR PEER REVIEW 6 of 11

Egg Abundance and Ratio According to Morphology (Normal and Abnormal)
The abundance of calanoid eggs ranged from 0.69 to 1.49 × 10 6 eggs m −2 during August and 0.59 to 1.08 × 10 6 eggs m −2 during April (Figure 5).The egg abundance was >1.00 × 10 6 eggs m −2 in August (Station 2, 3) and April (Station 1), and tended to increase towards the inner side of the bay during April.The normal and abnormal eggs were distinguished based on the egg envelope features and content.During August, the proportion of abnormal eggs decreased from stations 1 to 4, and the occurrence rate of normal eggs at station 4 was approximately 90% (Figure 6a).Similarly, during April, the proportion of abnormal eggs decreased from stations 1 to 4, but their occurrence rate was lower than that in August (Figure 6b).The normal and abnormal eggs were distinguished based on the egg envelope features and content.During August, the proportion of abnormal eggs decreased from stations 1 to 4, and the occurrence rate of normal eggs at station 4 was approximately 90% (Figure 6a).Similarly, during April, the proportion of abnormal eggs decreased from stations 1 to 4, but their occurrence rate was lower than that in August (Figure 6b).
Water 2021, 13, x FOR PEER REVIEW 6 of 11

Egg Abundance and Ratio According to Morphology (Normal and Abnormal)
The abundance of calanoid eggs ranged from 0.69 to 1.49 × 10 6 eggs m −2 during August and 0.59 to 1.08 × 10 6 eggs m −2 during April (Figure 5).The egg abundance was >1.00 × 10 6 eggs m −2 in August (Station 2, 3) and April (Station 1), and tended to increase towards the inner side of the bay during April.The normal and abnormal eggs were distinguished based on the egg envelope features and content.During August, the proportion of abnormal eggs decreased from stations 1 to 4, and the occurrence rate of normal eggs at station 4 was approximately 90% (Figure 6a).Similarly, during April, the proportion of abnormal eggs decreased from stations 1 to 4, but their occurrence rate was lower than that in August (Figure 6b).

Discussion
The number of eggs found in the collected sediment should be regarded as the minimum value, because damage or loss can occur during the sampling process, and repeated sampling at the same station is required to provide reliable quantitative data.The egg abundance (0.59-1.49× 10 6 eggs m −2 ) was similar to the previously reported range (10 3 -10 7 eggs m −2 ) for marine and estuarine calanoid copepods (Table 1) [16,[32][33][34][35].In Masan Bay, Acartia omorii, Calanus sinicus, and Centropages abdominalis account for over 60% of the copepod population during spring and winter, and A. erythraea, Pseudoiaptomus marinus, and Paracalanus parvus account for over 70% during summer and autumn [36].
Although copepod eggs were not identified at the species level, a significant number of eggs in Masan Bay was presumed to originate from the copepods A. omorii; P. parvus; C. abdominalis; A. erythraea; and C. sinicus [36][37][38].However, the eggs of all species did not appear in the sediment.The representative species was P. parvus.This heterogeneity is prominent in estuaries and coasts, but eggs have not been reported to be found in sediments [39].Marcus [40] reported that eggs cannot withstand sediment abrasion because they are fragile.In addition, P. parvus does not appear to produce dormant eggs [41].Therefore, the eggs from this species were not likely to have contributed to the eggs in the sediment, even in this study.The diameter of Calanus sinicus eggs (165-175 µm) is approximately double the size of Acartia and Centropages eggs [42].No large eggs were found in this study.Therefore, we assumed that most of the eggs observed were from A. omorii, A. erythraea, and C. abdominalis, due to their size and presence in the sediment.The semi-closed Masan Bay has a slow current velocity (<10 cm s −1 ) in the inner bay (Station 1-3), and the mean residence time of seawater is very long (54 d); thus, the exchange with offshore water is slow [43,44].The sedimentation rates can reach 1.3-2.0cm year −1 [45].Therefore, the redistribution of eggs in the sediment by the current appears to be limited.In addition, this study showed that most eggs in the surface sediment (top 0.5 cm) of all vertices spawned within one year by local females present in the water column, and then sunk.The sinking rates of marine copepod eggs reportedly range from 15 to 35 m −1 day −1 [46,47], and almost all eggs sink to the bottom before hatching in shallow waters with hypoxic and anoxic sediments [6,46].As the studied site has a depth of <20 m, it is likely that a significant portion of the eggs sank into the sediment before hatching.In this study, the proportion of morphologically abnormal eggs was significantly higher in August (under hypoxic conditions) than in April (under normoxic conditions).Several studies have reported that exposure to low oxygen (<0.16 mL L −1 ) or anoxic conditions causes quiescence in the subitaneous eggs of marine copepods [17,18,[50][51][52].The short exposure (≤11 d) to low oxygen or anoxic conditions does not significantly affect the viability of quiescent eggs, as they resume hatching when reincubated in normoxic seawater [18,[50][51][52].However, as the exposure time increases, the hatching and viability of the eggs decreases.For example, Invidia et al. [18] reported that the exposure to low oxygen for ≥15 d caused a significant reduction in hatching and a strong reduction in the life expectancy of Acartia tonsa eggs.Although some eggs survived exposure for four weeks or more, due to anaerobic metabolism, many eggs of three calanoid copepods (Acartia tonsa, Centropages hamatus, and Labidocera aestiva) died within two to four weeks of exposure to anoxia [15,51].Long exposure (≥60 days) to anoxia can impair the viability of eggs [17].
Diapause is a pivotal point for resurrection ecology that relies upon the confined area-resilience of communities during the resting stages [53,54].Belmonte and Rubino [54] reported that resurrection ecology can explain the emergence of species according to the seasons, many of which primarily depend on bottom sediments in a confined area.The identified species are involved in this phenomenon.Diapause eggs have devices (spines) to avoid sinking to anoxic layers before the completion of the first embryonic stages [55][56][57].Delayed sinking may allow early embryonic development to complete and the egg to enter the resting phase before reaching the anoxic bottom [54].Hypoxic conditions are not suitable for embryonic development or egg hatching, and resting eggs can wait until the oxygen concentration suitable for hatching is restored, depending on the season [50,54].However, in this study, we focused on the quiescence of eggs.A large number of diapause eggs exist in the sediments at the bottom of Masan Bay as a strategy to maintain the population.
In this study, the eggs that sank in the sediments at the inner stations (Station 1-2) during summer (August 2011) were exposed to higher sulphide concentrations and lower pH under hypoxic conditions than the eggs that sank in the sediments at the middle and outer stations (Station 3-4).Studies have reported that the combination of sulphide and low pH can exert a greater effect on the viability of copepod eggs than low oxygen alone [17,18,58].Acartia tonsa eggs that were exposed to anoxia alone could still hatch after the 240 d incubation period, while eggs exposed to anoxia-sulphide at concentrations ranging from 10 µM to 10 mM did not hatch, thus indicating a limited tolerance [17].Additionally, Nielsen et al. [17] reported that sulphide could pass through the eggshell, which can impair the developmental capability of eggs as it increases their metabolic rate.In an experiment combining pH with anoxia-sulphide (~1 mM) conditions, the hatching rate of A. tonsa eggs exposed to pH 8.2 was significantly higher than that of eggs exposed to pH 6.5 [18].In addition, after 32 d of exposure, the hatching rate of eggs cultured at pH 8.2 ranged from 20% to 80%; however, the hatching of eggs cultured at pH 6.5 was almost completely suppressed.Invidia et al. [18] reported that, when a low pH is combined with anoxia-sulphide, the survival time of eggs can be shortened.Unlike the laboratory studies mentioned above, Choi et al. [58] compared the in situ hatching success of copepod eggs by installing plankton emergence trap/chambers (PET chambers) in the seabed-surface sediment at hypoxic and normoxic sites, and their results showed that the cumulative hatching rate in the hypoxic site (oxygen concentration: 0.6 mL L −1 , sulphide concentration: >300 µM, pH 7.3) was only 4%, which was much lower than the 57% at the normoxic site.
The lower abundance of eggs in the samples collected in spring, compared with the samples collected in summer, was likely the main reason for the hatching success during April.However, during August, the summer season, the conditions were hypoxic and contributed to the poorest conditions for successful hatching.During early spring, we expected much greater phytoplankton availability as there were no predators during the winter season, and the phytoplankton availability increased with spring mixing.Jónasdóttir and T. Kiørboe [59] reported that hatching success was influenced by the fertility of females and the nutritional quality of the food.Santhanam et al. [60] confirmed that the food concentration affected the egg production and hatching success in P. parvus.Huntley et al. [61] confirmed the importance of food quality in determining the rate of survival and development of Calanus pacificus nauplii and adults.Although this study did not consider the effect of food availability on females, it is unlikely that the food concentration acted as a limiting factor of egg hatchability, as the study area was an area with an average Chl-a concentration of 4.0 or higher from the spring to winter months [62].Moreover, these studies indicate that food quality affects maternal fertility, and the survival and development of offspring, and its effect on the fertility and viability of eggs is unknown [60][61][62].Future studies should consider a long-term survey into the relationship between nutrient conditions and phytoplankton availability, and copepod egg production and hatchability.

Figure 2 .
Figure 2. Normal and abnormal copepod eggs from the sediment of Masan Bay in August 2011 and April 2012.

Figure 4 .
The pH varied from 7.6 to 8.1 (August) and 7.8 to 8.1 (April), and was particularly low (<7.8) in the inner bay (St. 1,

Figure 2 .
Figure 2. Normal and abnormal copepod eggs from the sediment of Masan Bay in August 2011 and April 2012.

Figure 2 .
Figure 2. Normal and abnormal copepod eggs from the sediment of Masan Bay in August 2011 and April 2012.

Figure 4 .
The pH varied from 7.6 to 8.1 (August) and 7.8 to 8.1 (April), and was particularly low (<7.8) in the inner bay (St. 1,

Figure 4 .
The pH varied from 7.6 to 8.1 (August) and 7.8 to 8.1 (April), and was particularly low (<7.8) in the inner bay (St. 1, 2) during August (Figure 4a,b).The sediment pH tended to significantly decrease from the outer bay to the inner bay during both August and April.Water 2021, 13, 3116 5 of 11 the outer bay to the inner bay during both August and April.

Figure 5 .
Figure 5. Copepod egg abundance in the sediment of Masan Bay in August 2011 and April 2012 (data are presented as the mean ± standard error).

Figure 6 .
Figure 6.Relative composition of normal and abnormal eggs in the sediment of Masan Bay.(a) August 2011, and (b) April 2012.

Figure 5 .
Figure 5. Copepod egg abundance in the sediment of Masan Bay in August 2011 and April 2012 (data are presented as the mean ± standard error).

Figure 5 .
Figure 5. Copepod egg abundance in the sediment of Masan Bay in August 2011 and April 2012 (data are presented as the mean ± standard error).

Figure 6 .
Figure 6.Relative composition of normal and abnormal eggs in the sediment of Masan Bay.(a) August 2011, and (b) April 2012.

Figure 6 .
Figure 6.Relative composition of normal and abnormal eggs in the sediment of Masan Bay.(a) August 2011, and (b) April 2012.

Table 1 .
Summary of the abundance of copepod eggs in various locations.