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Short-term toxicity to fish

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Reference
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2012-02-07 to 2012-06-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: SEPA, The Guidelines for the Testing of Chemicals, 2004, 203 Fish Acute Toxicity Test.
Version / remarks:
2004
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Range finding test: Two nominal concentrations of 40mg/L and 300mg/L (arithmetic mean measured concentration were 21.8 mg/L and 197 mg/L, respectively).
Limit test: nominal concentration of 300mg/L (arithmetic mean measured concentration was 223mg/L) .
For the analysis of the actual concentration, 2ml of test solution was taken from each test vessel at the beginning, after 48 hours and at the end of the test. The samples were analyzed directly without storage.
Vehicle:
no
Details on test solutions:
Test solutions preparation:
The test substance was dissolved in test water to prepare the stock solution of 10g/L (mean measured concentration was 10.4g/L in limit test). A peristaltic pump system continuously dispensed the stock solution and test water to the mixing chambers. The stock solution used in limit test was not renewed.

In the range-finding test, the flow rates of stock solution and test water were 0.056ml/min and 13.84ml/min in 40mg/L treatment, respectively. The flow rates of stock solution and test water were 0.42ml/min and 13.48ml/min in 300mg/L treatment, respectively. The flow rate of test water in blank control was 14ml/min.

In the limit test, the flow rates of stock solution and test water were 0.42ml/min and 13.48ml/min in 300mg/L treatment, respectively. The flow rate of test water in blank control was 14ml/min.

Range-finding test
Two nominal concentrations of 40mg/L and 300mg/L (arithmetic mean measured concentration were 21.8mg/L and 197mg/L, respectively) were used in range-finding test. In this test, a blank control of test water was used.

Based on the results of the range-finding test, a limit test was performed. In this test, a blank control of test water and a nominal concentration of 300mg/L (arithmetic mean measured concentration was 223mg/L) were used.

Test organisms (species):
other: Gobiocypris rarus
Details on test organisms:
The test was performed with Gobiocypris rarus which were obtained from Institute of Hydrobiology, Chinese Academy of Sciences (ID: GR-IHB-111102). This freshwater fish species is suitable for fish toxicity testing because of their ease of culturing and handling, sensitivity to a variety of chemical substances, and the extensive data base for this species.

Before used in the test, all fish had been held in the laboratory under the following conditions for more than 14 days:
Water: the test water, tap water dechlorinated by activated carbon and sterilized by UV lamp was used.;
Temperature: 21 to 25℃;
pH: 6.0 to 8.5;
Oxygen concentration: > 80% of air saturation value (ASV);
Photoperiod: Light/Dark=12 hours/12 hours;
Feeding: fish were fed daily until 24 hours before the test was started.


The criterion of fish selection:
Length was 2.6±0.1cm (Mean±SD) and weight was 0.27±0.08g (Mean±SD) at the end of the test;
No diseases were observed during the acclimating period;
No mortality was present within 7 days before start of test.
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Post exposure observation period:
None
Hardness:
Total hardness: 165mg CaCO3/L
Test temperature:
The range of temperature in the test solutions and blank control was between 21.6 and 23.1°C
pH:
The range of pH value in the test solutions and blank control were between 7.00~7.88
Dissolved oxygen:
During the test period, the range of the dissolved oxygen concentrations in the test solutions and blank control were between 78.8~95.0%ASV.
Salinity:
Not applicable
Nominal and measured concentrations:
Range finding test: Two nominal concentrations of 40mg/L and 300mg/L (arithmetic mean measured concentration were 21.8mg/L and 197mg/L, respectively).
Limit test: nominal concentration of 300mg/L (arithmetic mean measured concentration was 223mg/L) .
Details on test conditions:
Water
The dechlorinated tap water, by activated carbon and sterilized by UV lamp, was used. The water was aerated continuously over 24h. Water quality parameters were as following:
Total hardness: 165mg CaCO3/L;
Oxygen concentration: >90% of ASV.

Test vessels:
5L glass tanks were used.

Conditions of exposure:
Duration: 96h
Test method: flow-through and frequency of water removal during the test was 20 litres per day
Loading: about 0.7g fish/litre
Temperature: 21.6~23.1℃
Photoperiod: Light: Dark=12 hours:12 hours
Oxygen concentration: 78.8~95.0 %ASV
Feeding: none

Range-finding test:

Five fish were introduced into each concentration and the test duration was 96 hours. Fish were observed and observation for dead fish was recorded at 0, 24, 48, 72 and 96 hours after test beginning. For the analysis of the actual concentration, 2ml of test solution was taken from each test vessel at the beginning, 48h and at the end of the test.

Limit test:

Ten fish were introduced into each concentration and the test duration was 96 hours. The fish were inspected at 0, 24, 48, 72 and 96 hours after test beginning and mortalities were recorded, if applicable. At the same time, the pH, dissolved oxygen and temperature were measured and recorded. For the analysis of the actual concentration, 2ml of test solution was taken from each test vessel at the beginning, after 48 hours and at the end of the test.
At the the end of the test, the body length and body weight of the test fish were compared to the respective results of the control group fish. The loading rate was also calculated with the body weight at the same time.

Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 223 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: Variation: -5.38 to +4.04%
Details on results:
The actual concentration of test solutions was determined by HPLC analysis method. The limit of detection (LOD) for the test substance in test medium was calculated to be 0.100 mg/L (based on a signal/ noise of 3.10). The routine limit of quantification (LOQ) for the test substance was calculated to be 0.500 mg/L (based on a signal/noise of 14.2). It was found that the average recovery was 85.8% and RSD (relative standard deviation) value was 3.98% for the spiked 5.00mg/L sample. In limit test, the measured concentration of test substance varied between -5.38%-4.04%.

In the range-finding test, there were no mortalities and abnormalities observed in fish of control group and treatment group during the test.

In the limit test, the arithmetic mean measured concentration of test substance was 223mg/L. The concentration of the stock solution was stable and it was not renewed in the limit test. See 19.1. There were no mortalities and abnormalities observed in fish of control group and treatment group during the test.

During the test period, the range of the dissolved oxygen concentrations in the test solutions and blank control were between 78.8~95.0%ASV. The range of temperature in the test solutions and blank control were between 21.6~23.1°C. The range of pH value in the test solutions and blank control were between 7.00~7.88.
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
None
Sublethal observations / clinical signs:

Method validation:

-     No mortality in the control at the end of the test

-     Exposure conditions were maintained constant throughout the test

-     The dissolved oxygen concentration in test solutions were between 78.8~95.0% ASV, which was greater than 60.0% of the air saturation value (ASV) throughout the test

-     During the test, the measured concentration of the test substance varied less than 20%, which were between -5.38%-4.04%

The test fulfilled the above requirements and was therefore considered to be valid.

The measured concentrations of the test substance in limit test:

Group

Nominal concentration (mg/L)

Measured concentration (mg/L)

0h

48h

96h

Arthmetic mean measured concentration

Variation (%)

Blank Control

0

ND

ND

ND

-

-

Treatment Group

300

232

211

226

223

-5.38~4.04

Stock Solution

1

10000

10500

10500

10400

10433

-1.28-1.60

2

10300

10600

10400

LOD=0.100mg/L, “ND”: not detection, “-”, not available.

The mortalities and abnormalities in range-finding test (5 fish in each concentration):

Group

Duration (h)

Mortalities

Cumulative mortalities

Abnormalities

Blank Control

0

0

0

none

24

0

0

none

48

0

0

none

72

0

0

none

96

0

0

none

Treatment Group

(Nominal concentration)

40mg/L

0

0

0

none

24

0

0

none

48

0

0

none

72

0

0

none

96

0

0

none

300mg/L

0

0

0

none

24

0

0

none

48

0

0

none

72

0

0

none

96

0

0

none

The mortalities and abnormalities in limit test (10 fish in each concentration):

Group

Duration (h)

Mortalities

Cumulative mortalities

Abnormalities

Blank Control

0

0

0

none

24

0

0

none

48

0

0

none

72

0

0

none

96

0

0

none

Treatment Group (Nominal concentration 300mg/L)

0

0

0

none

24

0

0

none

48

0

0

none

72

0

0

none

96

0

0

none

Dissolved oxygen, pH and temperature in test solutions and the control of the limit test

 Parameter

Blank Control

Treatment Group

(Nominal concentration 300mg/L)

0h

Oxygen(%ASV)

95.0

94.4

Temperature(°C)

22.8

23.1

pH

7.00

7.07

24h

Oxygen(%ASV)

86.1

87.3

Temperature(°C)

22.6

22.3

pH

7.86

7.83

48h

Oxygen(%ASV)

84.0

78.8

Temperature(°C)

22.3

22.6

pH

7.61

7.45

72h

Oxygen(%ASV)

83.2

79.6

Temperature(°C)

21.8

21.6

pH

7.88

7.82

96h

Oxygen(%ASV)

85.7

87.7

Temperature(°C)

22.9

22.5

pH

7.55

7.56

Validity criteria fulfilled:
yes
Conclusions:
The test results showed that the 96-h LC50 of the test substance Reactive Yellow F01-0555 to Gobiocypris rarus was higher than 223 mg/L (arithmetic mean of measured concentration).
Executive summary:

In accordance with “SEPA, The Guildelines for the Testing of Chemicals, 2004, 203 Fish Acute Toxicity Test” and “OECD Guidelines for the Testing of Chemicals. 203 Fish, Acute Toxicity Test 1992”, the acute toxicity of the test substance Reactive Yellow F01-0555 to Gobiocypris rarus was determined by a flow-through test.

The test substance was dissolved in test water to prepare the stock solution of 10 g/L (mean measured concentration was 10.4 g/L in limit test). The test solutions were prepared by dilution of the stock solution with test water. Based on the results of range-finding test, a treatment group with arithmetic mean measured concentration of 223 mg/L and a blank control group were used in limit test.. The test duration was 96 h.

There were no mortalities and abnormalities observed in the treatment group during the test period. No mortality occurred in the control group during the test and the dissolved oxygen concentration was greater than 60.0 %of the air saturation value throughout the test duration. Hence, the test was considered to be valid. During the test, the measured concentrations of the test substance varied less than 20 %. The 96h LC50 of the test substance to Gobiocypris raruswas higher than 223 mg/L under tested conditions.

Description of key information

The test results showed that the 96-h LC50 of the test substance Reactive Yellow F01-0555 to Gobiocypris rarus was higher than 223 mg/L (arithmetic mean of measured concentration).

Key value for chemical safety assessment

Additional information