Registration Dossier

Administrative data

Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: HJ/T 153-2004, The guidelines for the testing of chemicals[S]. Beijing: SEPA, 2004
Deviations:
no
Qualifier:
according to
Guideline:
other: CRC-MEP. The Guidelines for the Testing of Chemicals, Effects on Biotic Sysmtem[M]. The 2nd edition. Beijing: China Enviroment Press. 2013:86-93
Deviations:
no
Qualifier:
according to
Guideline:
other: GB/T 21854-2008, Fish, Early-life Stage Toxicity Test, Beijing: SAC, 2008
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
gas
Details on test material:
- Purity: 99.9945%

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
-Concentrations Sampled: 10.0 mL water samples before and after renewal were taken (at least in duplicate) from each concentration during the test at 0, 6, 12, 18, 24, and 32 days.
-Sampling method: 10.0 mL water samples were drawn into 60 mL headspace bottles; the headspace bottles were placed in a water bat at 80±2°C for heating 30 minutes. Using a gas-tight syringe, 1 mL upper gas was drawn out from the headspace bottle, and manually injected into the gas chromatograph to determine the concentration

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test substance is a gas. According to the information provided by the sponsor, the water solubility of the test substance is 280 mg/L (25°C), so the test medium was prepared as a stock solution. An air cylinder containing the test substance was passed through a hose to 5 L of test water with a rate of 4 to 6 bubbles per second for 6 hours. This stock solution was colourless and clear. The stock solution was then further diluted as necessary, to provide the remaining test concentrations. Each test chamber contained 1 L of test solution covered by inert lids with zero headspace and solutions were renewed every 24 hours.
- Differential loading: 30 eggs/1 L
- Controls: One dilution medium control series was tested in addition to the test substance treatment series.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): The stock solution was colourless and clear.

Test organisms

Test organisms (species):
other: Rare Minnow (Gobiocypris rarus)
Details on test organisms:
TEST ORGANISM
- Common name: rare minnow
- Strain: Gobiocypris rarus
- Source: Parent individuals were healthy adult fish without surface damage from a breeding population in a recirculating system.
- Feeding during test:
Brood fish: brine shrimp
Newly-hatched larvae; Artemia nauplii (newly hatched)
Juveniles: Artemia nauplii (48-hours old)
Time to first feeding: 6 days after spawning

ACCLIMATION
- Acclimation period:14-day pretest
- Type and amount of food: fish were fed brine shrimp
- Feeding frequency during acclimation: twice daily

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
The male and female fish were paired at 1:1 ration in an incubator. When the male was observed chasing the female, eggs and sperm were collected from the fish and the eggs artificially inseminated to get fertilized eggs. Before the test, the fertilized eggs were taken at random under a microscope to confirm the batch of eggs were in the same cell stage.

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
32 d
Remarks on exposure duration:
Hatching period 4 d, post-hatch 28 d, a total of 32 d.
Post exposure observation period:
No

Test conditions

Hardness:
145 mg CaCO3/L
Test temperature:
25.0-25.2°C
pH:
7.67-7.86
Dissolved oxygen:
85-96%
Salinity:
Freshwater
Conductivity:
No data
Nominal and measured concentrations:
0.625, 1.25, 2.5, 5, and 10 mg/L (Nominal)
0.053, 0.131, 0.426, 0.960, and 1.53 mg/L (Analytical)
Details on test conditions:
The concentration of the test substance was demonstrated to be maintained within ±20% of the measured initial concentration throughout the test under a 24 h-renewal condition.

During the test, the following conditions were maintained:

Volume of the test solution: 1 L/test chamber
Loading: 30 eggs/L
Stocking densities: 60 eggs for each treatment and control, divided into 2 replicates
Replicates: two per test substance treatment and control
Duration: hatching period 4 d, post-hatch 28 d, a total of 32 d
Light: 16-hour light and 8-hour dark cycle daily
Temperature: 25.0°C - 25.2°C for rare minnow
Oxygen concentration: 85% - 96%
Feeding:
Brood fish: brine shrimp;
Newly-hatched larvae: Artemia nauplii (newly hatched);
Juveniles: Artemia nauplii (48 h old);
Time to first feeding: 6 days after spawning.

Overall survival of fertilized eggs and post-hatch success in the controls were over 90%. The study met the test guideline validity criteria (pH within the range of 6.0 - 8.5, dissolved oxygen concentration: not less than 60% of the air saturation value; temperature: not differing by more than ±1.5°C between test chambers; the survival of fertilized eggs in the controls not less than 66%, and the post-hatch success in the controls over 70%), therefore the test was valid.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): At the end of the test, individual lengths were measured with measurement software of an Asana microscope. Total length was used for length measurements. At the end of the test, all surviving fish were measured for dry weight (24 hours at 60°C) and individual weight was measured through weight loss method (Note: Weight loss method, the first step was to determine all larvae weights [m1], the second step was to remove a larva carefully with surgical tweezers, the third step was to weigh total remaining larvae weights [m2]. The individual larvae weight = m1-m2).

RANGE-FINDING STUDY: According to an acute toxicity test to fish (Gobiocypris rarus) conducted at the facility, the 96-hour LC50 was 1.78 mg/L (measured concentration). Therefore, the nominal concentrations of 0.625, 1.25, 2.5, 5, and 10% stock solutions were selected for the early-life stage toxicity test. A blank control was tested in parallel (dilution medium without test substance).
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
32 d
Dose descriptor:
EC10
Effect conc.:
0.762 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
other: hatching
Key result
Duration:
32 d
Dose descriptor:
EC10
Effect conc.:
0.262 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
other: survival
Key result
Duration:
32 d
Dose descriptor:
NOEC
Effect conc.:
0.131 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
other: length and weight
Key result
Duration:
32 d
Dose descriptor:
LOEC
Effect conc.:
0.426 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
other: length and weight
Details on results:
During the test period, the hatching rates recorded in control treatment and treatments exposed to the test substance (at 0.625, 1.25, 2.5, 5, and 10% stock solutions) ranged between 81.7 and 100%, and the survival rates between 0 and 98.3%. In addition, all surviving fish appeared normal. All fish were dead on the 11th day in the 10% stock solution treatment.
Results with reference substance (positive control):
No postive control was included in this study
Reported statistics and error estimates:
Based on analysis of variance, the lengths and weights data for each test substance concentration were compared with the control values using the Bartlett's test (ANOVA, STATA 10.0). The results showed that there was no significant difference in treatment groups (at 0.625%, 1.25% stock solutions) compared with the blank control group (p > 0.05). There was significant difference in treatment groups (at 2.5%, 5% stock solutions) compared with the blank control group (p < 0.05).

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
EC10 (hatching) = 0.762 mg/L (based on measured concentration);
EC10 (survival) = 0.262 mg/L (based on measured concentration);
NOEC (length & weight) = 1.25% stock solution (measured concentration 0.131 mg/L);
LOEC (length & weight) = 2.5% stock solution (measured concentration 0.426 mg/L).
Executive summary:

Under semi-static conditions with a 24 h-renewal, the effects on fish (Gobiocypris rarus) early-life stages exposed to the test substance were assessed according to the following guidelines: "The guidelines for the testing of chemicals" (HJ/T 153-2004); Procedure 210 of the "Guidelines for Testing of Chemicals" of the "OECD: Fish, Early-life Stage Toxicity Test" adopted on July 26, 2013.

 

During the test period, the pH values of the control and test solutions were between 7.67 and 7.86, the dissolved oxygen (DO) values varied from 85% to 96% of the air saturation at the test temperature, and the temperature of the test solutions was maintained at 25.0°C - 25.2°C. Overall survival of fertilized eggs and post-hatch success in the controls were over 90%. The study met the test guideline validity criteria (pH within the range of 6.0 - 8.5, dissolved oxygen concentration: not less than 60% of the air saturation value; temperature: not differing by more than ±1.5°C between test chambers; the survival of fertilized eggs in the controls not less than 66%, and the post-hatch success in the controls over 70%), therefore the test was valid.

 

In order to confirm the stability of the test substance in the test medium, samples were taken during the test period and the concentrations of the test substance were quantified by GC-ECD. As the results of the standard solution analysis, a linear regression equation was obtained with the peak area values vs. the concentration of the test substance (0.05, 0.10, 0.50, 1.00, 2.00 and 5.00 mg/L): A =421.88 c +33.805, with good linearity of r2 = 0.9998. The result showed that linearity for the test substance in the concentration range of 0.05 mg/L to 5.00 mg/L was good. The nominal concentrations of 0.625%, 1.25%, 2.5%, 5% and 10% stock solutions were used in the test. The analytical results showed that the measured concentrations were 0.053, 0.131, 0.426, 0.960 and 1.53 mg/L, respectively. The concentration of the test substance in the test solutions was stable throughout a period of 24 h (deviation within 20%), thus a semi-static test design with 24 h-renewal was reasonable.

 

The hatching rates recorded in control treatment and treatments exposed to the test substance (at 0.625%, 1.25%, 2.5%, 5% and 10% stock solutions) ranged between 81.7% and 100% and the survival rates between 0% and 98.3%. The survival fish appeared normal. All fish were dead on the 11th day in 10% stock solutions treatment. Based on analysis of variance, the lengths and weights data for each test substance concentration were compared with the control values using the Bartlett's test (ANOVA, STATA 10.0). The results showed that there was no significant difference in treatment groups (at 0.625%, 1.25% stock solutions) compared with the blank control group (p > 0.05). There was significant difference in treatment groups (at 2.5%, 5% stock solutions) compared with the blank control group (p < 0.05). Therefore, the results showed that under valid semi-static test conditions (24 h-renewal), the LOEC (lowest observed effect concentration) and the NOEC (No observed effect concentration) of the test substance to fish in an early-life stage test were as follows:

 

EC10 (hatching) = 0.762 mg/L (based on measured concentration);

EC10 (survival) = 0.262 mg/L (based on measured concentration);

NOEC (length & weight) = 1.25% stock solution (measured concentration 0.131 mg/L);

LOEC (length & weight) = 2.5% stock solution (measured concentration 0.426 mg/L).