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Long-term toxicity to fish

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Reference
Endpoint:
fish short-term toxicity test on embryo and sac-fry stages
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014 April 3 to 2014 August 8
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Different species of fresh -water fish not indigenous to Europe or North America
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 215 (Fish, Juvenile Growth Test)
Deviations:
yes
Remarks:
Test species different than OECD 215 recommendation.
GLP compliance:
yes
Specific details on test material used for the study:
Identifier: EXP1200078
Appearance: Very dark brown (almost black) viscous liquid
Batch: E00275-350
Sample Expiration Date: end-2014
Purity:100%
Analytical monitoring:
yes
Details on sampling:
The water samples of control treatment and highest concentration treatment (100 mg / L WAFs) were collected and measured when freshly prepared and immediately prior to renewal at the start of the study (0 d) and weekly thereafter (3, 9, 18, 28 d).
Measurements of pH, dissolved oxygen and temperature were carried out at the beginning and weekly thereafter (3, 9, 18, 28 d).
Vehicle:
no
Details on test solutions:
1. Preparation of standard stock solution
TC storage solution (TC = 2000 mg/L, TIC = 1000 mg/L, TOC = 1000 mg/L): dissolving 3.50 g NaHCO3, 4.42 g Na2CO3 and 2.13 g potassium acid phthalate into 1 L deionised water.

2. Working solution
Prepare the standard TC working solution of 1.00, 2.00, 4.00, 10.0, 20.0, 40.0, 100, 200 TC mg/L by drawing and diluting the above TC storage solutions with deionised water, and their TOC concentrations were 0.50, 1.00, 2.00, 5.00, 10.0, 20.0, 50.0, 100 mg/L (TOC) respectively.
Test organisms (species):
other: Gobiocypris rarus
Details on test organisms:
The test species of Rare minnow (Gobiocypris rarus, Batch No.: JF20140320, Hatch date: Mar. 20, 2014) were bred by
our own lab. A population of a single stock was used in the test. Details concerning the source of the fish were recorded in the raw data.

Feeding fry were held for 14 days in holding tanks supplied with a continuous flow of aerated water before being used for testing. During the pre-culture, the following conditions were maintained:

› Light: 16 hours photoperiod daily (light intensity: 1000 lux);
› Temperature: 22ºC to 23ºC for Rare minnow;
› Oxygen concentration: 90% of the air saturation;
› -Feeding: The fish were fed daily with live food (Artemia) daily and the quantity of food being kept constant 4% dry weight related to the initial fish weight (about 0.15g).
o 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.: JF20140320).

Using a general anaesthetic (an aqueous solution of 100 mg/L tricaine methane sulphonate (MS 222) neutralized by the
addition of two parts of sodium bicarbonate per part of MS 222), a subsample of 10 fishes before the test were weighed
individually as wet weights (blotted dry) with the precision of 1 mg. The wet-weight of the acclimated batch of test fish at the start of test is showed in Table 7. Handling of juvenile fish was done with the utmost care to avoid stressing and injuring test animals.
Test type:
semi-static
Water media type:
freshwater
Total exposure duration:
28 d
Remarks on exposure duration:
72h renewal
Hardness:
hardness of the test water was 122.2 mg (CaCO3)/L
Test temperature:
temperature: (23 ± 2) ºC
pH:
pH was 7.55
Dissolved oxygen:
oxygen concentration was about 98% of the air saturation value
Salinity:
not mentioned
Details on test conditions:
Details on test conditions described in attached Tables 1 through 4
Key result
Duration:
28 d
Dose descriptor:
other: LOEL
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
28 d
Dose descriptor:
other: LOEL
Effect conc.:
> 1.55 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: Total Organic Carbon (TOC)
Basis for effect:
growth rate
Key result
Duration:
28 d
Dose descriptor:
other: NOEL
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
28 d
Dose descriptor:
other: NOEL
Effect conc.:
> 1.55 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: Total Organic Carbon (TOC)
Basis for effect:
growth rate
Details on results:
Results including body weights and growth rates are presented in attached Tables 5 through 7
Reported statistics and error estimates:
Reported in attached Table 8

Calibration curve and representative chromatograms as attached Figures 01 - 06.

Validity criteria fulfilled:
yes
Conclusions:
Under conditions of the testing, there is no impact from EXP1200078 on juvenile fish growth.
Executive summary:

The results showed that under valid semi-static test conditions (72 h-renewal), no fish died in control and treatments groups. Fish in all treatments and control groups were observed alive and appeared normal. In the 28d test period, the LOEL (lowest observed effect loading rate) and the NOEC (No observed effect loading rate) of the test substance to juvenile rare minnow were concluded as follows:

28 d-LOEL >100 mg/L WAFs (measured concentration 1.55 mg/L);

28 d-NOEL = 100 mg/L WAFs (measured concentration 1.55 mg/L).

Description of key information

The results showed that under valid semi-static test conditions (72 h-renewal), no fish died in control and treatments groups. Fish in all treatments and control groups were observed alive and appeared normal. In the 28d test period, the LOEL (lowest observed effect loading rate) and the NOEC (No observed effect loading rate) of the test substance to juvenile rare minnow were concluded as follows:

28 d-LOEL >100 mg/L WAFs (measured concentration 1.55 mg/L);

28 d-NOEL = 100 mg/L WAFs (measured concentration 1.55 mg/L).

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater fish:
100 mg/L

Additional information

Under conditions of the testing, there is no impact from EXP1200078 on juvenile fish growth when conducted in accordance with OECD Guideline 215.