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Long-term toxicity to fish

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Reference
Endpoint:
fish, juvenile growth test
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: - Not all details available on analytics (raw data). - No detailed information on test organism (source, culture etc.).
Qualifier:
according to
Guideline:
OECD Guideline 215 (Fish, Juvenile Growth Test)
Deviations:
yes
Remarks:
- Not all details available on analytics (raw data). - No detailed information on test organism (source, culture etc.).
GLP compliance:
yes
Remarks:
GLP issued by State Environmental Protection Administration (SEPA) of the People’s Republic of China
Analytical monitoring:
yes
Details on sampling:
Samples were taken from all test solutions when freshly prepared (the fresh test media) and immediately prior to renewal (the aged test media) at the start of the study (0 d) and weekly thereafter (3, 9, 18, 28 d). The highest and lowest test concentrations were analyzed after after certain pre-treatments.
Pre-treatment method of the sample: 10 mL of the water samples collected were diluted by 2-fold with methanol and filtrated by 0.22 µm membrane.
Vehicle:
no
Details on test solutions:
The test solution of M-DIOL was prepared by directly adding appropriate amount of the test item into test water. Ultrasonication for 30 min was used to facilitate the dispersion of the test substance.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
The test species of Zebra fish, Brachydanio rerio, were obtained as fry from a commercial fish supplier of Guangzhou Hongfa Aquatic Animals Culture Company in China. A population of a single stock was used in the test. Details concerning the source of the fish were recorded in the raw data (Batch No. JF20111120).
Feeding fry were held for 14 days in holding tanks supplied with a continuous flow of aerated water before being used for testing.

-Feeding: The fish were fed daily with live food (Artemia) daily and the quantity of food being kept constant 2% to 4% dry weight related to the initial fish weight. Food was withheld from the stock population for 24 h prior to the start of the test.
During the holding period the tanks was inspected daily and any debris or unhealthy or dead fish were removed. Fish received no treatment for disease in the two weeks preceding the test and during the test.
After the 48 h settling-in period, no mortality was observed in the following 7 days. So this batch of fish was accepted (Batch No. JF20111120).
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
28 d
Test temperature:
22 to 23 °C
pH:
7.5 to 7.9
Dissolved oxygen:
95% to 105% of the air saturation
Nominal and measured concentrations:
Nominal: 20, 50, 80, 100, 150 mg/L
Measured: 20 - 20.4 mg/L; 150 - 154 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Tanks made of chemically inert material, with a sealable inert lid, and with a capacity of approximately 3 L
- Type (delete if not applicable): open / closed
- Material, size, headspace, fill volume:
- Aeration:
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate):
- No. of organisms per vessel:
- No. of vessels per concentration (replicates):
- No. of vessels per control (replicates):
- No. of vessels per vehicle control (replicates):
- Biomass loading rate: 0.1 to 1.0 g of fish/L. At the start of the test, fishes weighed about 0.063 g (± 7.08%) in average.
- Light: 16-hour light and 8-hour dark cycle daily (light intensity: 1000 to 1500 lux).
- Feeding: daily (the quantity of food being kept constant at 4% dry weight related to the initial fish weight).

TEST MEDIUM
Good quality tap water which had been dechlorinated for at least 24 h was used. The total hardness of the test water was 122.2 mg (CaCO3)/L, oxygen concentration was about 98% of the air saturation value and pH was 7.55 at room temperature. Characteristics of the dilution water are measured at least twice a year by Jiangsu Provincial Center for Disease Prevention and Control.
Reference substance (positive control):
not specified
Duration:
28 d
Dose descriptor:
EC50
Effect conc.:
> 150 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
>= 150 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
>= 150 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
In all treatments and the control, fishes were observed alive and appeared normal.
Reported statistics and error estimates:
Based on the analysis of variance (ANOVA SPSS 17.0), the pseudo specific growth rates for each concentration were compared with the control values using an multiple comparison method of Dunnett’s tests.
Validity criteria fulfilled:
yes
Conclusions:
The study was performed under Chinese GLP. This study result, with some restrictions, can be used for risk assessment and classification and labelling.
Executive summary:

Under semi-static conditions of 72 h-renewal, the effect on growth rates and other observed effects in juvenile fish(Brachydanio rerio) exposed to the test substance for 28 days was conducted according to the following guidelines:“The guidelines for the testing of chemicals”, SEPA(HJ/T 153 -2004); Procedure 215 of the “Guidelines for Testing of Chemicals”of the OECD: " Fish, Juvenile Growth Test” (2000) etc.

During the whole test period, the pH values of the control and Test Media were between 7.5 and 7.9, and the Dissolved Oxygen (DO) values varied from 95% to 105% of the air saturation at the test temperature, and the temperature of the Test Media was maintained in the range of 22oC to 23oC. All fishes in the control group were normal. The mean weight of fish in the controls increased more than 60%. So the study met the acceptability criteria prescribed by the protocol (dissolved oxygen concentration: no less than 60% of the air saturation value; temperature:(23±2)oC and not differ by more than ±1oC between test chambers; the increasing rate of the mean wet-weight of fish: no less than 50% of the initial weight). Therefore the test was considered valid.

In order to confirm the stability of the test item in the test medium, concentrations of the test samples during the test were analyzed by UPLC-PDA. A linear regression equation was obtained between the concentration and the response (Peak area): y= 47142.9x+ 1615.2, with good linearity of r2=0.99996, where y represents the response (Peak area); and x is the concentration of the test item (mg/L).The results show that linearity for the concentration range of 0.50 mg/L to 20.0 mg/L is good. Besides, the minimum detection amount of UPLC-PDA for M-DIOL is 2.5×10-9g, and the minimum detection concentration for water sample is 0.50 mg/L.

The results showed that under valid semi-static test conditions (72 h-renewal), the LOEC, NOEC and the EC50 for the growth rate are all greater than 150 mg/L (measured concentration 155 mg/L).

Description of key information

The results showed that under valid semi-static test conditions (72 h-renewal), the LOEC, NOEC and the EC50for the growth rate are all greater than 150 mg/L (measured concentration 155 mg/L).

Key value for chemical safety assessment

Additional information