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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:
supporting study
Study period:
From 7 February 2000 to 10 March 2000
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: See "Remarks"
Remarks:
This study was performed according to international guidelines with GLP statement. All validity criteria were fulfilled. However, the analytical method used in this study is not adapted (the limit of quantification is too low: 1 mg/L), very small test substance concentrations were found during the test. Furthermore, mortality was 0 or 100% at the end of the definitive test, the 96h-LC50 calculated as the geometric mean of the highest concentration without observed effect and the LC100 is not reliable.
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
no
Principles of method if other than guideline:
Not applicable.
GLP compliance:
yes (incl. certificate)
Remarks:
2000-01-18
Analytical monitoring:
yes
Vehicle:
no
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Remarks on exposure duration:
none
Duration:
96 h
Dose descriptor:
other: The highest concentration without observed effect
Effect conc.:
0.89 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
1.78 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
1.26 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: Since at all concentrations, mortality was 0 or 100% at the end of the test, the 96h LC50 was calculated as the geometric mean of the highest concentration without observed effect and the LC100

Table 6.1.1/2: Analytical measurements

Nominal (mg/L)

Measured (mg/L)

Geometric mean (mg/L)

0h

23.9h

24h

47.9h

48h

71.9h

72h

96h

5

1.57

BLQ

1.40

1.09

BLQ

BLQ

2.21

BLQ

0.89

10

3.90

0.449

3.71

1.63

1.83

0.635

4.62

1.81

1.78

Number in italic are values below the limit of quantification used for data interpretation.

Water quality parameters:

Temperature: between 14.5 and 15.5°C.

pH: between 7.57 to 8.12.

Oxygen: between 6.2 to 10 mg/L.

Table 6.1.1/3: Cumulative mortality in definitive test

Time (hours)

Mortality at each nominal concentration (mg/L)

Control

0.313

0.625

1.25

2.5

5

10

0

2

4

24

48

72

96

0/8

0/8

0/8

0/8

0/8

0/8

0/8

0/8

0/8

0/8

0/8

0/8

0/8

0/8

0/8

0/8

0/8

0/8

0/8

0/8

0/8

0/8

0/8

0/8

0/8

0/8

0/8

0/8

0/8

0/8

0/8

0/8

0/8

0/8

0/8

0/8

0/8

0/8

0/8

0/8

0/8

0/8

0/8

0/8

0/8

0/8

3/8

3/8

8/8

Validity criteria fulfilled:
yes
Conclusions:
Under experimental conditions: The highest concentration resulting in no mortality at 96 hours was 0.89 mg/L; the lowest concentration resulting in 100% mortality at 96 hours (LC100) was 1.78 mg/L. Since at all concentrations, mortality was 0 or 100% at the end of the test, the 96h-LC50 was calculated as the geometric mean of the highest concentration without observed effect and the LC100, i.e. 1.26 mg/L.
Executive summary:

This study was performed according to international guidelines with GLP statement, to assess the acute toxicity of the test substance in the rainbow trout Oncorhynchus mykiss, using a 96h semi-static test. The test solutions were changed after 24, 48 and 72 hours. Mortality and sub-lethal effects at each concentration were recorded at 0, 2, 4, 24, 48, 72 and 96 hours.

A preliminary test which included a limit (at 10 mg/L) and a range-finding test (at 0.01, 0.1 and 1 mg/L) preceded the definitive test. The results show that in the limit test, mortality was 0% in the control and 100% in the limit test solution (10 mg/L, nominal) at the end of the 96 hour; and in the range-finding test, no mortality was observed at all concentrations, up to 1 mg/L, at the end of the 96 hour test.

In the definitive test, six concentrations of the test substance and one control were used at nominal concentrations of 0, 0.313, 0.625, 1.25, 2.5, 5 and 10 mg/L. Eight fish were exposed to each concentration for 96 hours under semi-static renewal conditions. No mortalities were observed at any of the concentrations of 0 through 5 mg/L (nominal) at the end of the 96 hours. For the 10 mg/L (nominal), 3 of the fish died after 48 hours and by the end of 96 hours, all 8 fish were dead. No sub-lethal effects were observed at 1.25 mg/L (nominal) or below.

Chemical analysis was undertaken with HPLC/UV to measure the concentration of the test substance in the solutions of the definitive test at 5 and 10 mg/L. Other definitive test solutions (0.313 to 2.5 mg/L) were not analyzed because some or all measured concentrations would have been below the limit of quantification (1 mg/L). Except for one value, measured concentrations in the fresh test solution (T0, T24, T48 and T72 hours) for the nominal 10 mg/L rate were in the range 3.71 to 4.62 mg/L. On the other hand, all measured concentrations in the 24 hour test solution (T23.9, T47.9, T71.9 or T96 hours) at the 10 mg/L rate were significantly lower than the corresponding initial values at T0, T24, T48 or T72 hours (<80%). According, all toxicity effects were therefore based on the geometric mean of measured concentrations for the solution at the nominal 10 mg/L rate. Results for the nominal 5 mg/L rate similarly considerably less than the theoretical value. In fact, some values measured in the solution at the 5 mg/L rate could not be used since they were markedly below half the limit of quantification (LOQ = 1 mg/L). Therefore, a theoretical geometric mean corresponding to half that of the 10 mg/L solution (calculated from measured concentrations) was used. Geometric mean concentrations were calculated as 0.89 and 1.78 mg/L for the test solutions at 5 and 10 mg/L rates, respectively.

Under experimental conditions:

- The highest concentration resulting in no mortality at 96 hours was 0.89 mg/L.

- The lowest concentration resulting in 100% mortality at 96 hours (LC100) was 1.78 mg/L.

- Since at all concentrations, mortality was 0 or 100% at the end of the test, the 96h-LC50 was calculated as the geometric mean of the highest concentration without observed effect and the LC100, i.e. 1.26 mg/L.

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2003-08-05 to 2003-11-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This study was performed according to OECD Guideline n° 203 with GLP statement. All validity criteria were fulfilled and no deviations were observed.
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
Principles of method if other than guideline:
not applicalbe.
GLP compliance:
yes (incl. certificate)
Remarks:
2003-02-13
Analytical monitoring:
yes
Details on sampling:
- Concentrations:1.0, 1.8, 3.2, 5.6, and 10 mg/L
- Sampling method: Fish, in groups of 10, were exposed to different concentrations of test material for 96 hours. The number of mortalities and any adverse reactions to exposure in each test and control vessel were determined 3 and 6 hours after the start of exposure and then daily throughout the study until termination of the experiment.
- Sample storage conditions before analysis: Duplicate samples were taken and stored frozen (approximately -20°C) for further analysis if necessary.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Based on the results of the range-finding tests and the preliminary recovery and stability analyses conducted, the test material solutions for the definitive test were prepared by stirring an excess (100 mg/L) of the test material in dechlorinated tap water at approx. 2000 rpm at a temperature of 14°C for 24h. After 24 hours, undissolved test material was removed by filtration (0.2 µm) to give a saturated solution of the test material of 10 mg/L which was then further diluted
- Eluate: Lab tap water
- Differential loading: cf. method description.
- Controls: identical conditions but not exposed to the test material.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no data

Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: rainbow trout
- Strain: no data
- Source: Brow Well Fisheries Limited (Yorkshire, UK)
- Age at study initiation (mean and range, SD): no data
- Length at study end (length definition, mean, range and SD): 5.0 cm (SD 0.2)
- Weight at study end (mean and range, SD): 1.81 g (SD 0.15)
- Method of breeding: in a glass fibre tank with a single pass water renewal system
- Feeding during test: no

ACCLIMATION
- Acclimation period: 14 days of acclimation
- Acclimation conditions (same as test or not): yes
- Type and amount of food: commercial trout pellets
- Feeding frequency: no data
- Health during acclimation (any mortality observed): no mortality
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
no data
Hardness:
100 mg/L as CaCO3
Test temperature:
14.3-14.9°C
pH:
7.5-7.6
Dissolved oxygen:
>= 8.8 mg O2/L
Salinity:
not applicable.
Nominal and measured concentrations:
- nominal conc.: 1.0, 1.8, 3.2, 5.6, 10 mg/L
- measured conc.: See table 6.1.1/2 in "Any other information on results incl. tables".
Details on test conditions:
TEST SYSTEM
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 22L
- Aeration: no auxiliary aeration
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): not applicable
Based on the mean weight value this gave a loading rate of 0.82 g bodyweight/litre.
A semi-static test regime was employed in the test involving a daily renewal of the test preparations.

TEST MEDIUM / WATER PARAMETERS
Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener) giving water with a total hardness of approximately 100 mg/l as CaC03. After dechlorination and softening the water was passed through a series of computer controlled plate heat exchangers to achieve the required temperature.

OTHER TEST CONDITIONS
- Photoperiod: 16h light / 8h 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.

TEST CONCENTRATIONS
- Range finding study: Yes
- Test concentrations: control; 0.10, 1.0, 10 mg/L
- Results used to determine the conditions for the definitive study: The results showed no mortalities at the test concentrations of 0.10 and 1.0 mg/l. However, mortalities were observed at 10 mg/l.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
1.6 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 1.3-1.8
Duration:
96 h
Dose descriptor:
other: The highest concentration without observed effect
Effect conc.:
0.51 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
no data
Results with reference substance (positive control):
not applicable.
Reported statistics and error estimates:
no data

Table 6.1.1/1: cumulative mortality data in the definitive test.

Nominal concentration (mg/L)

Cumulative mortality (n=10)

% mortality

3 h

6 h

24 h

48 h

72 h

96 h

96 h

Control

0

0

0

0

0

0

0

1.0

0

0

0

0

0

0

0

1.8

0

0

0

0

0

0

0

3.2

0

0

0

1

4

5

50

5.6

0

0

0

10

10

10

100

10

0

10

10

10

10

10

100

Table 6.1.1/2: Analytical measurements

Sample

Nominal concentration (mg/L)

Concentration found (mg/L)

Expressed as a percent of the nominal concentration (%)

0 hours

Control

1.0

1.8

3.2

5.6

10

<LOQ

0.919

1.74

2.93

5.36

9.82

-

92

97

92

96

98

24 hours

Old media

Control

1.0

1.8

3.2

5.6

10

<LOQ

0.135

0.325

0.171 / 0.0786*

0.346 / 0.235*

8.22

-

13

18

5 / 2*

6 / 4*

82

24 hours

Fresh media

Control

1.0

1.8

3.2

5.6

10

<LOQ

0.904

1.56

2.88

5.56

8.67

-

90

87

90

99

87

48 hours

Old media

Control

1.0

1.8

3.2

5.6

0.0163 / <LOQ*

0.218

0.320

0.630

1.48

-

22

18

20

26

48 hours

Fresh media

Control

1.0

1.8

3.2

10

<LOQ

1.06

1.90

3.17

13.6

-

106

105

99

136

72 hours

Old media

Control

1.0

1.8

3.2

<LOQ

0.258

0.459

0.882

-

26

25

28

72 hours

Fresh media

Control

1.0

1.8

3.2

10

0.0196*

1.06

1.80

3.46

10.8

-

106

100

108

108

96 hours

Old media

Control

1.0

1.8

3.2

<LOQ

0.260

0.535

1.46

-

26

30

46

* Duplicate samples, stored frozen prior to analysis.

Validity criteria fulfilled:
yes
Conclusions:
The LC50(96h), based on the time-weighted mean measured test concentrations of the test media, was 1.6 mg/L with 95% confidence limits of 1.3 -1.8 mg/L. The highest concentration without observed effect was 0.51 mg/L.
Executive summary:

A study was performed to assess the acute toxicity of the test material to rainbow trout. The method followed that described in the OECD Guidelines 203, under GLP.

Following a preliminary range-finding test, fish were exposed (n = 10), to aqueous solutions of the test material over a range of nominal concentrations of 1.0, 1.8, 3.2, 5.6, and 10 mg/L for a period of 96 -h, under semi-static test conditions. 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 -h.

Chemical analysis of the freshly prepared test media sampled at 0, 24, 48, 72 hours showed measured concentrations of between 87% and 108% of the nominal test concentrations. However, analysis of the old or expired test media sampled at 24, 48, 72, 96 hours showed a marked decline in the measured test concentrations which were shown to range from 2% to 46% of the nominal values with the exception of the 10 mg/L test concentration at 24 hours which showed a measured value of 82% of nominal.

Given this decline in measured test concentrations it was considered justifiable to base the results on the time-weighted mean measured test concentrations of the test media to give a "worst case" analysis of the data.

The LC50(96h), based on the time-weighted mean measured test concentrations of the test media, was 1.6 mg/L with 95% confidence limits of 1.3 -1.8 mg/L. The highest concentration without observed effect was 0.51 mg/L.

Description of key information

OECD Guideline 203, GLP, key study, validity 1:

96h-LC50 (Oncorhynchus mykiss) = 1.6 mg/L (95% confidence limits of 1.3 -1.8 mg/L) based on time-weighted mean measured concentrations.

Key value for chemical safety assessment

LC50 for freshwater fish:
1.6 mg/L

Additional information

To assess the acute toxicity of the substance in rainbow trout Oncorhynchus mykiss, two studies are available, in accordance to international guidelines with GLP statement.

The first study, performed by SafePharm laboratory, was considered valid. Following a preliminary range-finding test, fish were exposed to aqueous solutions of the test material over a range of nominal concentrations of 1.0, 1.8, 3.2, 5.6, and 10 mg/L for a period of 96 -h, under semi-static test conditions. Chemical analysis of the old or expired test media sampled at 24, 48, 72, 96 hours showed a marked decline in the measured test concentrations and so it was considered justifiable to base the results on the time-weighted mean measured test concentrations of the test media to give a "worst case" analysis of the data. 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 -h. The LC50(96h), based on the time-weighted mean measured test concentrations of the test media, was 1.6 mg/L with 95% confidence limits of 1.3 -1.8 mg/L. The highest concentration without observed effect was 0.51 mg/L.

The second study, performed by CIT laboratory, was not considered reliable based on chemical analysis and the choice of test concentrations. An HPLC/UV method was used with a limit of quantification of 1 mg/L. Since at all concentrations, mortality was 0 or 100% at the end of the test, the 96h-LC50 was calculated as the geometric mean of the highest concentration without observed effect and the LC100, i.e.1.26 mg/L.