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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:
Experimental Starting Date: 12 June 2012. Experimental Completion Date: 20 July 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study performed according to GLP and guideline
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
GLP compliance:
yes (incl. certificate)
Remarks:
Date of inspection: 19-21 July 2011. Date of signature: 31 August 2011
Analytical monitoring:
yes
Details on sampling:
Water samples were taken from the control and all surviving test groups at 0 (fresh media), 24 (old media), 72 (fresh media) and 96 hours (old media) for quantitative analysis. The samples were stored at approximately -20° C prior to analysis.

Duplicate samples, and samples at 24 (fresh media), 48 (old and fresh media) and 72 hours (old media) and the 100% v/v saturated solution after 100% mortality at that level, were taken and stored at approximately -20 °C for further analysis if necessary.
Details on test solutions:
For the purpose of the range finding and definitive tests the test item was prepared as a saturated solutions.

Amounts of test item were separately added to dechlorinated tap water respectively and stirred using a propeller stirrer at approximately 1500 rpm for 24 hours. After stirring, any undissolved test item was removed by filtration through a 0.2 µm Gelman AcroCap filter and the filtrates pooled to give 100 % v/v saturated solution. Aliquots of the 100 % v/v saturated solution were each separately diluted in a final volume of 10 liters of dechlorinated tap water, and stirred using a flat bladed mixer for approximately 1 minute.
Test organisms (species):
Pimephales promelas
Details on test organisms:
TEST ORGANISM
- Common name: Fathead minnow
- Source: Fish were obtained from Osage Catfisheries Inc., Osage Beach, Missouri, USA
- Length at end of study: Mean standard length of 2.9 cm (sd = 0.1)
- Weight at end of study: Mean weight of 0.36 g (sd = 0.07)

ACCLIMATION
Fish were maintained in a glass fiber tank with a "single pass" water renewal system. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. The water temperature was controlled at 20° C to 21°C with a dissolved oxygen content of greater than or equal to 8.5 mg O2/L. The stock fish were fed ZM 400 fish food which was discontinued approximately 48 hours prior to the start of the definitive test. There was zero mortality in the 7 days prior to the start of the test.


Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Test temperature:
20°C to 21°C
pH:
7.5 to 7.9
Dissolved oxygen:
Dissolved oxygen concentration expressed as a percentage of Air Saturation Value (ASV)= 56 to 99 %
Nominal and measured concentrations:
Range finding test: 0, 1.0, 10, 100 % v/v saturated solutions
Definitive test: 10, 18, 32, 56 and 100% v/v saturated solutions
Details on test conditions:
TEST SYSTEM
- Test vessel: 10 liter glass exposure vessels were used for each test concentration. The test vessels were covered to reduce evaporation. Test vessels were not aerated.
- Aeration: No
- Renewal rate of test solution: Daily renewal
- No. of organisms per vessel: Range finding test: 3. Defintive test: 10

TEST MEDIUM / WATER PARAMETERS
The test water used for both the range-finding and definitive tests was the same as that used to maintain the stock fish. The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the test.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 96 hours

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Any mortalities and sub-lethal effects of exposure were recorded at 3, 6, 24, 48, 72 and 96 hours after the start of exposure. The criteria of death were taken to be the absence of both respiratory movement and response to physical stimulation
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
4.4 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95% CL: 1.6 - 12 mg/L
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
12 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
1.6 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
Range finding test:
Chemical analysis of the 10 and 100% v/v saturated solution at 0 hours showed measured concentrations of 1.49 and 18.6 mg/L respectively. Analysis of the corresponding old media at 24 hours showed measured concentrations of 0.087 mg/L and 11.1 mg/L respectively.

All fish died with 100 % treatment. The results showed no mortalities at the test concentrations of 1.0 and 10 % v/v saturated solution.

Definitive test:
Verification of test concentrations:
Analysis of the freshly prepared 56% v/v saturated solution at 0 and 72 hours showed measured concentrations of 8.92 and 7.69 mg/L. Analysis of the corresponding old media at 24 and 96 hours showed measured concentrations of 0.087 mg/L. Analysis of the 100% v/v saturated solution at 0 (fresh media) and 24 hours (old media) showed measured concentrations of 17.3 and 7.75 mg/L respectively.

The time-weighted mean measured test concentrations for the 56 and 100% v/v saturated solution were calculated to be 1.6 and 12 mg/L respectively.

By 24 hours, all fish were dead or moribund in the 12 mg/L treatment group. No other mortalities were recorded in any other treatment group throughout the 96 hour test.

Cumulative Mortality Data in the Definitive Test:

Nominal

Concentration

(% v/v Saturated Solution)

Cumulative Mortality (Initial Population = 10)

%

Mortality

3
Hours

6
Hours

24 Hours

48 Hours

72 Hours

96 Hours

96
Hours

Control

0

0

0

0

0

0

0

10

0

0

0

0

0

0

0

18

0

0

0

0

0

0

0

32

0

0

0

0

0

0

0

56 (1.6 mg/L)

0

0

0

0

0

0

0

100 (12 mg/L)

1

1

10

10

10

10

100

Conclusions:
The acute toxicity of the test item to the freshwater fish fathead minnow (Pimephales promelas) has been investigated. Given the decline in measured test concentrations it was considered justifiable to base the results on the time-weighted mean measured test concentrations. The 96-Hour LC50 value of 4.4 mg/L with 95% confidence limits of 1.6 - 12 mg/L. The Lowest Observed Effect Concentration was considered to be 12 mg/L. The No Observed Effect Concentration was 1.6 mg/L.

Description of key information

Key study performed according to GLP and OECD Guideline 203:

The acute toxicity of the test item to the freshwater fish fathead minnow (Pimephales promelas) has been investigated. Given the decline in measured test concentrations it was considered justifiable to base the results on the time-weighted mean measured test concentrations. The 96-Hour LC50 value of 4.4 mg/L with 95% confidence limits of 1.6 - 12 mg/L.  The Lowest Observed Effect Concentration was considered to be 12 mg/L.  The No Observed Effect Concentration was 1.6 mg/L.

Key value for chemical safety assessment

LC50 for freshwater fish:
4.4 mg/L

Additional information

Introduction

A study was performed to assess the acute toxicity of the test item to fathead minnow (Pimephales promelas). The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No 203, "Fish, Acute Toxicity Test" referenced as Method C.1 of Commission Regulation (EC) No.440/2008 and US EPA Draft Ecological Effects Test Guidelines OPPTS 850.1075 "Fish Acute Toxicity Test, Freshwater and Marine".

 

Methods

Following a preliminary range-finding test, fish were exposed, in groups of ten, to an aqueous solution of the test item over a range of concentrations of 10, 18, 32, 56 and 100% v/v saturated solution for a period of 96 hours at a temperature of 20°C to 21°C under semi-static test conditions. The test item solution was prepared by stirring an excess (50 mg/L) of test item in test medium using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test item was removed by filtration (0.2 μm Gelman AcroCap filter, with the first approximate 500 mL discarded in order to pre-condition the filter) to produce a 100% v/v saturated solution of the test item. Media preparation trials indicated a water solubility value of approximately 22 mg/L when using this method of preparation. Dilutions of the 100% v/v saturated solution were prepared in test medium to give the remainder of the test series. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 3 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours.

 

Results

The 96-Hour LC50 based on the time-weighted mean measured test concentrations was 4.4 mg/L with 95% confidence limits of 1.6 - 12 mg/L. The Lowest Observed Effect Concentration was considered to be 12 mg/L. The No Observed Effect Concentration was 1.6 mg/L.

Analysis of the test preparations was conducted at the No Observed Effect Concentration (NOEC) and above only. Analysis of the freshly prepared 56% v/v saturated solution at 0 and 72 hours showed measured concentrations of 8.92 and 7.69 mg/L. Analysis of the corresponding old media at 24 and 96 hours showed measured concentrations of less than the limit of quantitation (LOQ) of the analytical method (assessed as 0.087 mg/L). Analysis of the 100% v/v saturated solution at 0 (fresh media) and 24 hours (old media) showed measured concentrations of 17.3 and 7.75 mg/L respectively.

 

Given this decline in measured test concentrations it was considered justifiable to base the results on the time-weighted mean measured test concentrations to give a "worst case" analysis of the data. The time-weighted mean measured test concentrations for the 56 and 100% v/v saturated solution were calculated to be 1.6 and 12 mg/L respectively.