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Long-term toxicity to fish

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Reference
Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Deviations:
not specified
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Idle operation was performed before starting exposure, and the concentrations of the test liquid at all of the test concentrations confirmed. The test liquid at all the test concentrations was analyzed at least once a week during the exposure period (including at the end of exposure). Test liquid was sampled from one vessel at every test concentration and analyzed using high-performance liquid chromatography (HPLC). Samples for measurement were taken for every analysis from a different sampling vessel at each test concentration.
Vehicle:
no
Details on test solutions:
Preparation of the Test Liquid
Test stock liquid and water for the test were successively mixed at a constant flow rate using a flow-through test apparatus, and the test liquid was supplied to each test vessel with a pump for supplying test liquid. Prior to exposure, it was confirmed that there was no variation in the quantity of test liquid supplied to each test vessel at the same test concentration.
During the exposure period, the flow rates of test stock liquid and water for the test were measured at least once a week, and it was confirmed that there was no fluctuation of ≥10% with respect to the set value. Moreover, the quantity of test liquid supplied was measured in at least one vessel at every test concentration at least once a week, confirming that a flow rate (water exchange rate) corresponding to at least 5 times the capacity of the test vessel per 24 hours was maintained.
The method for preparing the test stock liquid is presented below. The test stock liquid was to be used within 13 days of preparation. The test stock liquid was confirmed to be stable for 13 days at room temperature.

Preparation of the test substance stock liquid
Amount of test substance taken --> 50.0 g
Solvent --> Ultrapure water
Final volume --> 2.0 L
Container --> Screw cap bottle
Test substance concentration --> 25,000 mg/L
Mixing method --> Mixing by manual stirring
Test organisms (species):
Oryzias latipes
Details on test organisms:
Test Organism
Common name: Medaka
Scientific name: Oryzias latipes
Developmental stage: Fertilized eggs before the morula stage
Rearing condition of the parent fish for obtaining fertilized eggs: Healthy and macroscopically normal individuals were selected and reared, and the resulting fertilized eggs were used in the study.
Supplier of parent fish: Reared in-house
Date started rearing (Date eggs collected): 29 August 2019
Lot number of parent fish: 18-M-0829B
Age of parent fish: Within 1 year after hatching
Rearing water: Dechlorinated tap water
Rearing method: flow through
Water temperature: 24±1°C
Dissolved oxygen conc'n: ≥80% of saturated dissolved oxygen concentration
Lighting: Indoor lighting, 16 hr light (≤1000 lux)/8 hr darkness
Feed: Live feed (Vietnamese brine shrimp eggs acquired from Fujimoto Taiyodo KK): newly hatched brine shrimp larvae
Amount of feed: to satiation (1-2 times/day)
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
45 d
Hardness:
46-48 mg CaCO3/L on starting exposure and 36-42 mg CaCO3/L at the end of the exposure
Test temperature:
test concentrations: 25.1-25.9°C
control: 25.2-25.9°C
pH:
6.9-7.4
Dissolved oxygen:
7.1-8.6 mg/L; throughout the period of exposure the dissolved oxygen concentration was ≥60% of the saturated concentration
Salinity:
n/a
Nominal and measured concentrations:
The measured mean values for concentrations 1-5 were 0.616, 1.26, 2.47, 4.95 and 10.1 mg/L
Nominal: 0.65, 1.3, 2.5, 5.0, 10.0 mg/L
Details on test conditions:
Test Vessels and Flow-Through Apparatus, etc.
Test vessels, etc. were sterilized before use.
1) Test Vessels: 3.0-L glass water tanks (frontage 120 x width 200 x height 140 mm). A container fitted with a fine stainless steel screen on two sides was suspended in said water tank before hatching, and the embryos were exposed therein.
2) Flow-through apparatus: A drainpipe for drainage, fitted with a fine stainless steel screen, was attached to the 3.0-L glass water tanks and the test liquid was supplied using a pump for test water, a pump for test stock liquid and a pump for supplying test liquid, as described on the next page. Test water and test stock liquid were respectively supplied to a mixing bottle in fixed quantities by using the respective pumps, stirred with a stirrer, and then supplied dropwise to each of the test vessels by using a pump for supplying test liquid.

Test Conditions
1) Duration of exposure: 45 days
2) Exposure method: Flow-through system
3) Volume of test liquid: 43 L/day per vessel, volume of water in the test vessels: ca. 2.5 L (water exchange rate: ca. 17 times/day)
4) Replications: 4 vessels/test concentration
5) No. of test organisms: 80 fish/test concentration (20 fish/vessel)
6) Housing density: ≤0.5 g/L per day, at all times ≤5 g/L
7) Water temperature: 25±2°C (water temperature within the range of the test temperature above during exposure period, with variation within test vessels and days not exceeding ±1.5°C)
8) Dissolved oxygen concentration: ≥60% of saturated concentration (no aeration)
9) pH: Not adjusted
10) Lighting: Indoor lighting, 16 hr illumination (≤1000 lux)/8 hr darkness
11) Feeding: BSN given 1-2 times a day to satiety for fish and juvenile fish

Preparation of the Test Liquid
Test stock liquid and water for the test were successively mixed at a constant flow rate using a flow-through test apparatus, and the test liquid was supplied to each test vessel with a pump for supplying test liquid. Prior to exposure, it was confirmed that there was no variation in the quantity of test liquid supplied to each test vessel at the same test concentration.
During the exposure period, the flow rates of test stock liquid and water for the test were measured at least once a week, and it was confirmed that there was no fluctuation of ≥10% with respect to the set value. Moreover, the quantity of test liquid supplied was measured in at least one vessel at every test concentration at least once a week, confirming that a flow rate (water exchange rate) corresponding to at least 5 times the capacity of the test vessel per 24 hours was maintained.
The test stock liquid was to be used within 13 days of preparation. The test stock liquid was confirmed to be stable for 13 days at room temperature.

Test Procedure
The mass of eggs taken from the parent fish was loosened and separated, and washed with water for the test. Then the necessary number of fertilized eggs at the same developmental stage were selected by microscope and used for the test. Twenty eggs were randomly introduced into each vessel, and exposure was started (one test concentration = 20 eggs x 4 vessels). Exposure was started at latest in the morula stage. The following items were monitored and measured.
1) Measurement of water quality and monitoring of the appearance of the test liquid
The temperature, dissolved oxygen concentration and pH, and appearance of the test liquid were monitored in all of the test vessels at least once a week during exposure period (including the beginning and end of exposure). The water temperature was measured every day in one control vessel. The total water hardness and salt concentration were measured at the beginning and end of exposure in one vessel at every test concentration.
2) Monitoring of the test organisms
(i) Eggs and embryos
• Survival, hatching and death: Monitored at least once a day, and the respective numbers recorded. Dead eggs and embryos were removed as soon as possible after observation. The criteria for judging death were as follows.
Eggs: Marked disappearance of semitransparency, becoming white and opaque.
Embryo: Absence of body movement and heartbeat.
• Developmental abnormality: Abnormality in the course of developmental was macroscopically monitored at least once a day, and the number was recorded. When abnormality was noted, the specific details were recorded. Sterilized vessel (petri dish, etc.) was used for sampling during the macroscopic observation.
(ii) Larval and juvenile fish
• Survival and death: Monitored at least once a day, and the respective numbers were recorded. Dead larval and juvenile fish were removed as soon as possible after observation. The criteria for judging death were as follows.
Larval and juvenile fish: Absence of movement, cessation of respiratory movement, absence of heartbeat, opacity of central nerves, lack of reaction to mechanical stimuli.
• Abnormalities: Malformation and behavioral disorders were monitored at least three times a week, and the numbers of abnormal larval and juvenile fish were recorded. Abnormal larval and juvenile fish were removed only when they were dead. The classification of behavioral disorder was as follows.
Incapable of swimming: Opercular movement only, cessation of movement such as swimming, or extreme abnormality, including rolling over or asphyxiation.
Abnormal swimming: Abnormal swimming behavior
Abnormal breathing: Abnormal opercular movement
• Body weight: Body weight (wet weight) of all the surviving fish was measured individually at the end of exposure.
• Full body length: Full body length of the surviving fish was measured at the end of exposure.
3) Observation of feeding and feeding status
BSN was given every day (1-2 times, excluding the end of exposure) to satiation, from the first day of hatching, and the quantity of feed was recorded. Because variation in the quantity of feed in each vessel at the same test concentration may cause a variation in the exposure concentration, the quantity of feed was the same for the same test concentrations. When the number of surviving fish in each vessel at the same test concentration differed, the quantity of feed was made the same as in the vessel at the same test concentration receiving the greatest quantity of feed.
Feeding status in each vessel was observed and recorded in each feed. Remaining feed and feces were removed with the necessary frequency.

Evaluation of the Results
Calculation of the Results
1) Items used in calculating the results
Results showing the following were calculated for each vessel.
• Hatching rate (%): No. hatched/no. of test organisms
• Survival rate after hatching (%): No. of survivors at the end of exposure/no. hatched
• Overall survival rate during the exposure period (%): No. of survivors at the end of exposure/no. of test organisms
• First hatching day and final hatching day
• Number of abnormal fish at the end of exposure
• Average body weight at the end of exposure
• Average full body length at the end of exposure
2) Concentrations of the test substance used for calculating results
The results were calculated based on the calculated mean measured values and the set values for test substance concentration
3) Results of observations
The results of observations, the cumulative hatching rate curve, the post-hatching survival rate curve and the overall survival rates during the exposure period obtained in the study were recorded in the report.
4) Calculation of the lowest observed effect concentrations (LOECs) and no observed effect concentrations (NOECs)
From the test results, the hatching rate, the survival rate curve after hatching, first hatching day and final hatching day, the number of abnormal fish at the end of exposure, the average body weight at the end of exposure and the average full body length at the end of exposure were determined in each vessel, and the LOEC and NOEC were determined in the control and each concentration and tested for significant differences using statistical techniques.
Key result
Duration:
45 d
Dose descriptor:
NOEC
Effect conc.:
>= 10.1 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
number hatched
Key result
Duration:
45 d
Dose descriptor:
NOEC
Effect conc.:
>= 10.1 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
45 d
Dose descriptor:
NOEC
Effect conc.:
>= 10.1 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
weight
Duration:
45 d
Dose descriptor:
NOEC
Effect conc.:
>= 10.1 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
length
Details on results:
1. Hatching rate
Lowest observed effect concentration (LOEC) : >10.1 mg/L
No observed effect concentration (NOEC) : ≥10.1 mg/L
2. Survival rate after hatching
Lowest observed effect concentration (LOEC) : >10.1 mg/L
No observed effect concentration (NOEC) : ≥10.1 mg/L
3. First hatching day
Lowest observed effect concentration (LOEC) : >10.1 mg/L
No observed effect concentration (NOEC) : ≥10.1 mg/L
4. Final hatching day
Lowest observed effect concentration (LOEC) : >10.1 mg/L
No observed effect concentration (NOEC) : ≥10.1 mg/L
5. Number of abnormal individuals at the end of exposure
Lowest observed effect concentration (LOEC) : >10.1 mg/L
No observed effect concentration (NOEC) : ≥10.1 mg/L
6. Average body weight at the end of exposure
Lowest observed effect concentration (LOEC) : >10.1 mg/L
No observed effect concentration (NOEC) : ≥10.1 mg/L
7. Average body length at the end of exposure
Lowest observed effect concentration (LOEC) : >10.1 mg/L
No observed effect concentration (NOEC) : ≥10.1 mg/L

Environmental Factors Which Might Have Affected the Reliability of the Results of The Study

Not applicable

Water Quality and External Appearance of the Test Liquid

The water temperature at all test concentrations was 25.1-25.9°C, and the water temperature in one vessel in the control each day during the test period was 25.2-25.9°C, which was within 25±2°C. The range of variation in water temperature between vessels and between days was maintained within ±1.5°C during the period of exposure. The dissolved oxygen concentration was 7.1-8.6 mg/L; throughout the period of exposure the dissolved oxygen concentration was ≥60% of the saturated concentration. The pH was 6.9-7.4. As far as the external appearance of the test liquid was concerned, no suspended solids, floating solids or precipitation were observed during the period of exposure at any test concentration, and the liquid was colorless. The total hardness was 46-48 mg CaCO3/L on starting exposure and 36-42 mg CaCO3/L at the end of the exposure, and the salt concentration was 0% both at the beginning and at the end of exposure.

Test Substance Concentration in the Test Liquid

The measured mean values for concentrations 1-5 were 0.616, 1.26, 2.47, 4.95 and 10.1 mg/L respectively. During the test period, the measured value as a percentage of the set concentration was 89-109%, maintained within ±20% of the set concentration during the period of exposure.

Hatching rate, First Hatching Day, Number Hatching Per Day and Final Hatching Day

The mean hatching rate in the controls was 93.8%, satisfying the hatching rate criterion for validity (>80%). The mean hatching rates at concentrations 1-5 were 98.8, 97.5, 100, 96.3 and 98.8%, respectively; no significant difference was observed compared to the controls.

The first hatching day in the controls was day 8. The first hatching day at concentrations 1-5 was day 7-9, 7-8, 7-8, 7-9 and 7-8, respectively; no significant difference was observed compared to the controls.

The final hatching day in the controls was day 16-20. The final hatching day at concentrations 1-5 was day 20-21, 12-23, 21, 18-21 and 10-21, respectively; no significant difference was observed compared to the controls.

Developmental Abnormalities in Eggs and Embryos

No developmental abnormality was observed in controls and concentrations 1, 3 and 5. Retarded development was observed in one individual at concentration 2; while at concentration 4, retarded development was observed in one individual and inactivity in two individuals.

Survival Rate

The survival rate after hatching and theoverall survival rate during the exposure periodare shown in Table 9. The survival rate of larval and juvenile fish after hatching is shown in Figure 3. The mean survival rate after hatching in controls was 92.3%, which satisfied the criterion for the validity of the study (survival rate after hatching in controls >80%). The mean survival rates after hatching at concentrations 1-5 were 96.3, 93.6, 95.0, 94.9 and 93.7%, respectively; no significant difference was observed compared to controls.The mean overall survival rate during the overall exposure periodin the controls was 86.3%, and at concentrations 1-5 the mean overall survival rates were 95.0, 91.3, 95.0, 91.3 and 92.5% respectively.

Abnormalitiesin Larval and Juvenile Fishand Feeding Status

No abnormal larval or juvenile fish were observed through to the end of exposure in the controls and concentrations 1-4. At concentration 5, a maximum of two wavy fish were observed from day 10 after starting exposure, and one wavy fish was observed at the end of exposure. Inability to swim was observed in one fish on days 35 and 38.

As far asfeeding status was concerned, no lowering of feeding ability was observed at any concentration.

Average Body Weight at the End of Exposure

The average body weight of the controls at the end of the exposure was 0.0687-0.0949 g. The average body weights at concentrations 1-5 were 0.0553-0.0806, 0.0640-0.0931, 0.0651-0.0854, 0.0906-0.0997 and 0.0781‑0.0977 g, respectively. The average body weights at the end of exposure at concentrations 4 and 5 were heavier than the average body weight of the controls, with the differencebeingstatistically significant. It was judged from this that the test substance does not inhibit the growth of medaka, and has no lethal or sublethal effect.

Average Full Body Length at the End of Exposure

The average full body length in the controls at the end of exposure was 17.78-19.36 mm. The average full body lengths at concentrations 1-5 were 16.06-18.38, 17.35-19.28, 17.06-19.15, 19.12-19.73 and 18.49-19.67 mm. No significant difference was observed at concentrations 1-5 compared to the controls.

Validity criteria fulfilled:
yes
Remarks:
The study was judged to be valid, since the results for water quality and appearance of the test liquid, hatching rate, first hatching day, number of hatching per day, final hatching day and survival rate satisfied the conditions for validity.

Description of key information

Acrylic acid did not cause adverse effects in a toxicity study according to OECD TG 210.

NOEC (hatching rate, survival rate, first/final hatching day, abnormal individuals, body weight/length; 45d, flow-through) >= 10.1 mg/L measured (highest test concentration)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
10.1 mg/L

Additional information

Acrylic acid was tested in a chronic toxicity test with Oryzias latipes under flow-through conditions. The study was conducted according to OECD TG 210 under consideration of GLP. Based on mean measured concentrations, the 45d NOEC (for all endpoints) was >= 10.1 mg/L. No effects could be recorded (LSI, 2019).