4. Discussion
These findings suggest that elevated CO2 affectsScophthalmus maximus more than previously reported. The effects of CO2 on aquatic organisms are diverse, and depend greatly on biotic factors such as species and life stage, and on abiotic factors such as exposure time and concentration (Fivelstad et al . 2018; Khan et al . 2018; Moran & Støttrup 2011; Mota et al . 2020; Noor et al . 2019; Pan et al . 2020).
At 7 d, relative to the control, plasma pH was significantly lower following CO2 treatment, whereas it increased over time, especially at 32 mg/L (Table 3). Under short-term exposure to elevated CO2, blood pH initially decreases; plasma HCO3− then increases to regulate acidity, returning pH to the initial level, or even higher (Pörtner et al . 2004). For example, in rainbow trout, elevated CO2 raised blood pH (Eddy et al . 1977). Throughout our experiment, blood pH was higher at 32 mg/L than at 8 mg/L. We speculate that compensatory regulation occurs in fish, and will study this further.
Here, fish growth responded negatively to elevated CO2, with the maximum CO2 level associated with the lowest SGR, WGR, and CF, and highest FCR. This is consistent with reports for other fish species, where long-term exposure to CO2concentrations of 5–40 mg/L negatively affected growth (Hhpa et al . 2020; Moran and Støttrup 2011; Mota et al . 2020; Neves and Brown 2015; Nmn et al . 2019; Stiller et al . 2015).
Elevated CO2 negatively impacted fish health. Cumulative survival declined significantly with increasing CO2, consistent with reports for other fish species (Huong et al . 2020; Hwang et al . 2009; Noor et al . 2021). Hb is the most important blood parameter for O2 delivery (Segneret al . 2012), and stressors can lead to insufficient Hb mobilization in the spleen and other hematopoietic organs, reducing Hb content (Harikrishnan et al . 2012). CO2 hampers oxygen-carrying capacity, via the “Bohr effect” (Bohr et al . 1904), whereby it causes H+ to bind to Hb, reducing its affinity for O2 and leading to inadequate O2 transport. In our study, at 32 mg/L, elevated plasma CO2 caused plasma Hb to be significantly lower than in the control. Similarly, elevated CO2 caused Hb to decrease in Salmo salar (Good et al . 2018).
Among the body’s molecules, erythrocyte Hb contains the most iron (Steinet al . 2010). The effects of CO2 on methemoglobinemia have not been reported. Here, methemoglobin levels were significantly higher at 32 mg/L than in the control. Elevated CO2 caused plasma Hb to increase slowly (Fig. 3A), reflecting a decline in Fe2+ levels. Iron homeostasis is regulated entirely by iron absorption via the digestive system. Fe2+ solubility decreases rapidly at pH > 6 (Stein et al . 2010). The blood pH levels that we observed reflect alkaline conditions (Table 3). We speculate that Fe2+ absorption declines rapidly in an alkaline intestinal environment. Reduced Hb, together with insufficient Fe2+ supplementation due to the alkaline intestinal environment, can lead to anemia, which may affect growth. Our results are consistent with this.
Fish and mammals have similar hepatic function, in which the liver accumulates, detoxifies, and metabolizes organic and inorganic pollutants (Köhler 1991). Because the liver responds rapidly to the external environment, hepatic pathological changes can be predictive biomarkers of aquatic toxicology (Frommel et al . 2012; Köhler 1990). Here, elevated CO2 led to hepatic damage, including the presence of fat vacuoles, atrophied nuclei, and unusual or necrotic hepatocytes. Under normal conditions, hepatocytes contain GOT and GPT. Hepatosteatosis causes transaminase levels to increase. Here, plasma transaminase levels were substantially elevated at 16, 24, and 32 mg/L CO2, reflecting liver damage (Gutiérrez & Solís 2009; Rezaeisaber & Nazer 2011; Zheng et al . 2006), consistent with our histology findings. Liver damage caused by high CO2 concentration has been reported for freshwater fish, and in isolated Antarctic fish hepatocytes (Good et al . 2010; Langenbuch & Pörtner 2004). In salmon, chronic stress caused by long-term elevated CO2 affects growth by modifying digestive capacity (Khan Jr. et al . 2006).
In fish, exposure to foreign substances (such as toxicants) causes fat to accumulate in the liver (White et al . 1973), which in turn raises HSI levels (Parikh et al . 2010). Here, fish condition initially improved, then declined over time under elevated CO2. We speculate that fat accumulates during early CO2 stress, leading to liver injury and causing nuclear atrophy, thereby reducing HSI levels.
Lysosomal dysfunction and destruction of the lysosomal vascular system causes fat vacuoles to appear in hepatocytes (Köhler 1990). We speculate that elevated CO2 disrupts and damages the lysosomes. Damage to the lysosomal system can impair normal hepatocyte function, leading to metabolic disorders, cell transformation, and cell death. The elevated GOT and GPT that we observed via histology and plasma analysis is consistent with this.
Fish blood biochemistry, including changes in plasma enzymes, can provide effective indicators of environmental stress, as well as an overview of the physiological state (Cole et al . 2001; Liet al . 2010; Noor et al . 2019). Under oxidative stress, antioxidant defense enzymes, such as SOD, CAT, and GPX, participate in clearing high-level ROS (Kochhann et al . 2009). SOD and CAT protect against oxidative damage by removing partially reduced oxygen species (Di Giulio et al .) Here, SOD, CAT, and GPX activity was significantly elevated at 32 mg/L CO2relative to the control, thereby alleviating hypoxia-induced stress. This proves that elevated CO2 affects the oxygen-carrying capacity of this species.
Lysozymes, important immune molecules in fish, are well known for their bactericidal effects (Panase 2017; Whang et al . 2011). They are also considered opsonins, activating both the complementary system and circulating phagocytes (Grinde 1989; Jollès & Jollès 1984). Further, some lysozymes have antiviral and anti-inflammatory activity (Ibrahimet al . 2001; Jollès & Jollès 1984; Lee et al . 1999; Samaranayake et al . 1997; Zhang et al . 2008). Here, exposure to 32 mg/L CO2 significantly enhanced lysozyme activity relative to the control, revealing that this defense mechanism was activated in in response to CO2 stress.
Stiller et al . (2015) have previously reported that CO2 negatively affects Scophthalmus maximusproduction and health, addressing two key parameters, WGR and SGR. Here, we have expanded this approach to include FCR, CF, HSI, plasma indices, histology, and quantitative gene expression. Even at CO2concentrations as low as 1–2 mg/L, O2 uptake, anti-predation behavior, and growth are reduced (Ou et al . 2015), and the olfactory system and central brain function are impaired (Porteus et al . 2018). However, dissolved CO2concentrations of 10–20 mg/L are commonly observed in commercial aquaculture systems (Gorle et al . 2018). For Atlantic salmon, based on simulations, CO2 levels <12 mg/L, with low stocking densities, did not negatively affect fish growth, physiology, or welfare (Mota et al . 2019). The effects of multiple stressors (such as temperature) and initial health status tend to be interactive, increasing the negative effects of exogenous substances on organisms (Almroth et al . 2019; Fivelstad et al . 2007; Park et al . 2020; Wendelaar 1997). Future studies of CO2 stress factors should therefore consider the combined effects of various biological variables.
The liver is the primary target organ of GH, which is synthesized in the pituitary gland, and is the primary source of IGF-1. In fish, the GH/IGF‑1 axis is a key endocrine pathway that regulates somatic-cell growth and development (Beckman 2011). In fish, GH regulates hepatic IGF-I release by binding to GHR (Bertucci et al . 2017; Di Prinzioet al . 2010; Reinecke et al . 2005). GH and IGF are easily disturbed by external factors, thereby affecting fish growth and development (Dang et al . 2018; De Las Heras et al . 2015; Yu et al . 2017). Using qRT-PCR, we therefore examined the expression of several key genes in the GH–IGF-1 axis, to evaluate how this species responds to elevated CO2 in aquaculture. GHR was significantly downregulated at 24 and 32 mg/L CO2. At the same time, elevated CO2significantly reduced IGF-1 and IGF-1R levels. We observed hepatic lesions and elevated GOT and GPT, which reflect CO2-induced liver damage that caused IGF-1 and IGF-1R levels to decrease. This is consistent with our growth analysis, providing further evidence that elevated CO2 inhibited growth, particularly at 32 mg/L.
Thyroid hormone, an important regulator of differentiation, growth, metabolism, and adaption to salinity (Craneet al . 2004; Orozco et al . 2002), binds to a receptor protein to regulate growth and metabolism. Here, this receptor gene was significantly downregulated. Although various toxicants and chemicals are thought to interfere with thyroid hormone and THR expression, reducing growth (Hu et al . 2020; Li et al . 2014; Wanget al . 2012), the effects of CO2 on THR have not previously been examined. We speculate that CO2 affects the liver and causes liver damage, which, in turn, inhibits growth.
In conclusion, 32 mg/L CO2 negatively affected juvenileScophthalmus maximus growth and health. Growth can be hampered even at CO2 concentrations below 8 mg/L. This provides insight into how juveniles of this commercially important marine fish respond to elevated CO2.