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Short-term toxicity to fish

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Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Qualifier:
according to
Guideline:
EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)
Qualifier:
according to
Guideline:
other: ASTM Standard E729 96: Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians
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-2013
Purity:100%
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Test solutions were prepared individually by adding a calculated amount of test substance into a tared glass beaker and sonicating for 60 minutes with a portion of the pre-measured 12 L of dilution water to be used for the final solution volume.

Following sonication, the contents of the beakers were emptied and mixed into the aspirator bottle containing the remaining 12 L of dilution water using a Teflon-lined stir bar for approximately 46 hours at a nominal WAF loading rate of 100 mg/L.

Care was taken to maintain the vortex depth for each WAF at approximately 30% of the test solution height.
Following the mixing period, the WAF solution was allowed to settle for approximately 1 hour and approximately
10 L of the test solution was decanted from mid-depth and filtered with a 20-25 µm paper filter.

Finally, the test solution was placed in the test chambers.

The negative control solution was dilution water only.
Test organisms (species):
Pimephales promelas
Details on test organisms:
The fathead minnow (Pimephales promelas) was selected as the test species for this study. Fathead minnows used in the test were received as juveniles from Chesapeake Cultures, Inc. of Hayes, Virginia. Identification of the species was verified by the supplier.

All fish used in the test were juveniles from the same source and year class, and the length of the longest fish measured was no more than twice the length of the shortest. The average total length of 10 negative control fish measured at the end of the test was 3.0 cm, with a range of 2.6 to 3.4 cm. The average wet weight (blotted dry) of 10 negative control fish measured at the end of the test was 0.19 grams, with a range of 0.12 to 0.25 grams. Loading was defined as the total wet weight of fish per liter of test water and was 0.19 g fish/L.

The fish were held for at least 14 days prior to the test in water from the same source and at approximately the same temperature as used during the test. During the 2 week period immediately preceding the test, water temperatures in the cultures ranged from 22.6 to 23.7¿C, measured with a liquid in-glass thermometer. The pH of the water ranged from 8.3 to 8.4, measured with a Thermo Orion Benchtop 4 Star Plus pH meter. Dissolved oxygen concentrations were ¿8.0 mg/L (¿92% of saturation), measured with a Thermo Orion Benchtop 3 Star Plus dissolved oxygen meter.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Hardness:
Hardness: 128 mg/L as CaCO3
Test temperature:
temperature during the study was 22 ± 1ºC
pH:
8.2 to 8.4 during the test
Dissolved oxygen:
Dissolved oxygen concentrations remained =6.9 mg/L (=80% of saturation) throughout the test.
Salinity:
no data
Details on test conditions:
Test Apparatus
Test chambers were 25 L stainless steel aquaria filled with approximately 10 L of test water. The depth of the test water in a representative test chamber was 14.3 cm. The chambers were indiscriminately positioned by replicate in a temperature-
controlled water bath to maintain the target water temperature throughout the test period. All test chambers were labeled
with the project number, test level and replicate designation.

Observations
All organisms were observed periodically to determine the number of mortalities in each treatment group. The numbers of individuals exhibiting signs of toxicity or abnormal behavior also were evaluated. Observations were made approximately 2,
24, 48, 72 and 96 hours after test initiation.

Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LL50
Remarks:
No observed mortality
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: No observed mortality
Key result
Duration:
96 h
Dose descriptor:
other: No-Mortality
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
96 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Reported statistics and error estimates:
Statistical Analyses
The use of a single limit level precluded the statistical calculation of LL50 values. Therefore, the 24, 48, 72 and 96-hour LL50 values, as well as the no-mortality level and the no-observed-effect level (NOEL), were determined by visual interpretation of the mortality and observation data.

Table 3

Cumulative Mortality and Observations

 

Nominal WAF

Loading Rate

(mg/L)

 

 

Rep.

 

No.

Exposed

2 Hours

 

24 Hours

 

48 Hours

No. Dead1

Observations2

 

No. Dead1

Observations2

 

No. Dead1

Observations2

 

 

 

 

 

 

 

 

 

 

 

Negative Control

A

10

0

10 AN

 

0

10 AN

 

0

10 AN

 

B

10

0

10 AN

 

0

10 AN

 

0

10 AN

 

C

10

0

10 AN

 

0

10 AN

 

0

10 AN

 

 

 

 

 

 

 

 

 

 

 

100

A

10

0

10 AN

 

0

10 AN

 

0

10 AN

 

B

10

0

10 AN

 

0

10 AN

 

0

10 AN

 

C

10

0

10 AN

 

0

10 AN

 

0

10 AN

 

 

 

 

 

 

 

 

 

 

 

1  Cumulative number of dead fish.

2  Observations:  AN = appear normal.


Table 3 (Continued)

Cumulative Mortality and Observations

 

Nominal WAF

Loading Rate

(mg/L)

 

 

Rep.

 

No.

Exposed

72 Hours

 

96 Hours

Cumulative

Percent

Mortality

No. Dead1

Observations2

 

No. Dead1

Observations2

 

 

 

 

 

 

 

 

 

Negative Control

A

10

0

10 AN

 

0

10 AN

0

 

B

10

0

10 AN

 

0

10 AN

 

 

C

10

0

10 AN

 

0

10 AN

 

 

 

 

 

 

 

 

 

 

100

A

10

0

10 AN

 

0

10 AN

0

 

B

10

0

10 AN

 

0

10 AN

 

 

C

10

0

10 AN

 

0

10 AN

 

 

 

 

 

 

 

 

 

 

1  Cumulative number of dead fish.

2  Observations:  AN = appear normal.

 

Table 4

LL50 Values

 

 

Time

LL50

(mg/L)

95% Confidence Interval

(mg/L)

Statistical

Method

24 Hours

> 100

--1

NA2

48 Hours

> 100

--1

NA2

72 Hours

> 100

--1

NA2

96 Hours

> 100

--1

NA2

1   95% confidence limits could not be calculated with the mortality data obtained.

2   NA = not applicable; the use of a single limit WAF loading rate precluded statistical calculation of an LL50 value.

 

Validity criteria fulfilled:
yes
Conclusions:
All fish in the negative control group and in the single treatment group appeared normal throughout the test, with no mortalities or overt signs of toxicity observed. The 96-hour LL50 value was >100 mg/L, the single limit WAF loading rate tested (Table 4, above). The no-mortality WAF loading rate and the NOEL were 100 mg/L.
Executive summary:

Fathead minnows (Pimephales promelas) were exposed for 96 hours under static conditions to a nominal limit WAF loading rate of EXP1200078 of 100 mg/L. The 96-hour LL50 value was >100 mg/L, the single limit level tested. The 96-hour no-mortality level and the NOEL were =100 mg/L.

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
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
Vehicle:
not specified
Details on test solutions:
Good quality tap water which had been dechlorinated for at least 24 hours 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.
Test organisms (species):
other: Gobiocypris rarus
Details on test organisms:
The test species of Rare minnow, Gobiocypris rarus (Batch No.: F20140215), were obtained as fry from a fish supplier of Institute of Hydrobiology, Chinese Academy of Sciences.

Feeding fry were at least held for 12 days in holding tanks supplied with a continuous flow of aerated water before being used for testing. In the study, the test fish were held 30 days in range-finding test and 43 days in definitive test, respectively. Fish to be used in the test was held for 7 days in water of the quality and temperature to be used in the test.

A photoperiod of 16 hours light, provided by overhead fluorescent tubes, and 8 hours dark was maintained. The oxygen concentration was more than 60% of the air saturation value.

The fish was fed daily during the holding period on proprietary fish food. They were held without food for approximately 24 hours before being placed into the test vessels.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Hardness:
- hardness of the test water was 122.2 mg (CaCO3)/L
Test temperature:
During the test, the temperature of the test mediums were maintained in the range of 22.7°C to 23.1°C,
pH:
pH was 7.55
Dissolved oxygen:
oxygen concentration was about 98% of the air saturation value
Salinity:
no data
Nominal and measured concentrations:
Nominal concentrations of 1.0, 10 and 100 mg/L were used in the range-finding test.

Nominal concentrations of 100 mg/L WAFs was used in the Limit Test.

Details on test conditions:
Range-finding Test
The range-finding test, carried out under static conditions, was regularly conducted to determine the range of concentrations for the subsequent test.

In the range-finding test, groups of fish (5 per group) were exposed to the test solutions with nominal concentration of 1.0, 10 and 100 mg/L. Synchronously one control group was conducted in test water without the test item. For each test tank 3 L
test solution was filled in. No replicates were used.

Limit Test
Static method was adopted in Limit Test. The stability of the test solution is confirmed by results indicated in Table 2 (deviation within 20%). Based on the results of the range-finding test, the concentration of 100 mg/L WAFs was assigned in Limit Test. Synchronously one control group was used in the test. 3 replicates were assigned for each treatment group and control group, while the initial number of testing fish was 7 for each group.

The test fish were randomly chosen and put in different test solutions after the temperature has been adjusted to the
required value. This was done in 30 min.

During the test, the following conditions were maintained:
-Light: 16 hours photoperiod daily (light intensity: 1000 lux to 1500 lux);
-Temperature: 22.7°C to 23.1°C;
-Oxygen concentration: 71 % to 79 % of the air saturation; No aeration.
-Feeding: none.


Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
There was no effect on either behaviour or mortality at any point during the test.
Results with reference substance (positive control):
With the conditions maintained as before, K2Cr2O7 was used as the test substance and the resulting 24 h-LC50 was
284 mg/L. The results of this study are shown in Table 4.

Mortality during theLimit Test

Nominal Conc.

(mg/L)

Measured Concentration(mg/L)

(no fitration)

Measured Concentration(mg/L)

(after fitration)

Initial Number

of Fish

The Number of the Dead Fish

24 h

48 h

72 h

96 h

Blank control

1.89¿

1.85

7

0

0

0

0

7

0

0

0

0

7

0

0

0

0

100 mg/LWAFs

1.52¿

ND¿

7

0

0

0

0

7

0

0

0

0

7

0

0

0

0

¿     The average of the TOC measured concentration at 0, 24, 48, 72, 96 h of control treatment, which was detected without filtration.

¿     The average of the measured concentration of test substance at 0, 24, 48, 72, 96 h of the test treatment, which was detected without filtration.

¿     Test substance concentration was low than the limit of quantification.

Visual Observations duringthe Limit Test

Nominal Concentration (mg/L)

Measured Concentration(mg/L)

(no fitration)

Measured Concentration(mg/L)

(after fitration)

Visual Observations

24 h

48 h

72 h

96 h

Blank control

1.89¿

1.85

NB¿

NB

NB

NB

NB

NB

NB

NB

NB

NB

NB

NB

100 mg/L WAFs

1.52¿

ND

NB

NB

NB

NB

NB

NB

NB

NB

NB

NB

NB

NB

¿The average of the TOC measured concentration at 0, 24, 48, 72, 96 h of control treatment, which was detected without filtration.

¿The average of the measured concentration of test substance at 0, 24, 48, 72, 96 h of the test treatment, which was detected without filtration.

¿Note:NB¿Normal behaviours

 

Validity criteria fulfilled:
yes
Conclusions:
The results showed that under valid static test conditions, the 96 h-LL50 of the test material to Rare minnow is greater than
the nominal concentration of 100 mg/L WAFs.
Executive summary:

Under conditions of the study, the Rare Minnow (Gobiocypris rarus), was tested using a WAF protocol for low solubility materials. There was no lethality or changes in behaviour at a limit concentration of 100 mg/L. Therefore, the acute LC50 was determined to be > 100 mg/L of the test substance.

Description of key information

A key GLP compliant study reporting 96h LL50> 100 mg/l loading rate WAF (OECD 203) . 

A supporting GLP compliant study reporting 96h LL50> 100mg/l loading rate WAF (OECD 203).

Key value for chemical safety assessment

LC50 for freshwater fish:
100 mg/L

Additional information

Two studies in GLP compliant studies reported the 96h LL50> 100 mg/l loading rate WAF.

Key study - fathead minnows (Pimephales promelas)

Supporting study - rare minnow (Gobiocypris raru)