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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:
23 March 2015 - 1 April 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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
Qualifier:
according to
Guideline:
other: SANCO/3029/99 rev.4 11/07/00: Residues: Guidance for generating and reporting methods of analysis in support of preregistration data requirements for Annex II (part A; Section 4) and Annex III (part A; Section 5) of directive 91/414
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Name of tested material (as cited in study report): Acilian (4-Amino-5-ethylsulfonyl-2-methoxybenzoic acid)
- batch No.of test material:HS00001
- Expiration date of the lot/batch:January 19, 2016
- Purity test date:February 12,2015
- Purity: 98.0 – 101.5% (nominal)
99.7% (analytical), according to certificate of analysis
0.1% water content

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:At room temperature (20 ± 5 °C), in the dark
- Stability under test conditions: yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Nominal:100 mg test item/L
- Sampling method:
Analysis of the Test Item Concentrations
Duplicate samples from the freshly prepared test media of the only test concentration and the control were taken at the start of the test and at day 3 (96 hours).All test medium samples were taken from the approximate centre of the aquaria.All samples were diluted with acetonitrile by a factor of 2.One additional sample of the blank control and the dilution solvent was taken at test start and test end without any sample treatment.
Two stock solutions were prepared. Approximately 50 mg of the test item were dissolved (2 minute ultrasonification, 91 minutes stirring) in 50 mL test water to obtain a stock solution of approximately 1 g test item/L. Appropriate amounts of these stock solutions were diluted with test water to obtain fortified samples at a level of 50 and 140 mg test item/L.

Standard Solutions used for the Quantification
Stock Solution:50.08 mg of the test item were dissolved in 50 mL acetonitrile to obtain a stock solution of approximately 1 g test item/L.
Standard Solutions: Appropriate amounts of the stock solution were diluted with acetonitrile / test water (1/1, v/v) to obtain standard solutions in the range from 10 to 80 mg test item/L.

Analysis of Treatment Samples and Control Samples
Sample Preparation:The samples were allowed to thaw to room temperature. They were then shaken well and treated with ultrasound for 1 minute to obtain homogenous samples. The samples were already diluted with acetonitrile by factor 2 directly after sampling.Fortified samples and analytical blank control samples: The samples were shaken well and were diluted with acetonitrile by factor 2.

- Sample storage conditions before analysis:All samples were stored in a freezer (≤ - 10 °C), protected from light until analysis was performed.

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
-Method:A test solution of nominal 100 mg/L was prepared by dissolving 1048.5, 1019.6, 1013 and 1012 mg of the test item homogeneously in 10.485, 10.196, 10.130 and 10.120 L test water by intense stirring for 2 hours and partial ultrasonic treatment for 5 minutes. The pH of the stock solution was adjusted to pH 8.0 ± 0.1 using 2 M NaOH. The test medium was prepared just before introduction of the test fish (= start of the test and test medium renewal on day 1, 2 and 3). Tested concentration was 100 mg/L test item.
- Controls:Reconstituted water (test water was used without addition of the test item)
- Differential loading:< 1 g fish/L test water
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No remarkable observations were made concerning the appearance of the test media.

Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name:Rainbow trout (Oncorhynchus mykiss)
- Source:Forellenzuchtbetrieb Störk, 88348 Bad Saulgau, Germany
- Age at study initiation : Juveniles,Male and female
- Length at study initiation : 5.05 cm ± 0.3 cm(Mean ± SD)
- Weight at study initiation :1.19 g ± 0.2 g (Mean ± SD)

ACCLIMATION
- Acclimation period:12 days,They were held in water of the quality to be used in the test for at least seven days immediately before testing.
- Acclimation conditions (same as test or not):No
Light: 16 hours photoperiod daily
Temperature: 13 - 17 °C
Oxygen concentration: at least 80 % of the air saturation value
- Feeding frequency during acclimation: yes, three times per week or daily until 24 hours before the test was started.
- Health during acclimation (any mortality observed):NO

FEEDING DURING THE TEST :none
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Hardness:
2.5 mmol/L (= 250.0 mg/L) as CaCO3
Test temperature:
14 to 15 °C
pH:
7.8 to 8.1
Dissolved oxygen:
93 to 100 % of the air saturation value during the test
Conductivity:
≤ 10 μScm-1
Nominal and measured concentrations:
Nominal:100 mg test item/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass aquaria
- Material, size, headspace, fill volume: Glass, 12 litre with 10 litre test medium
- Aeration: yes, slightly stirring
- No. of organisms per vessel:7
- No. of vessels per concentration (replicates):1
- No. of vessels per control (replicates):1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:ISO Medium was prepared according to OECD Test Guideline No 203
- Alkalinity:0.8 mmol/L
- Ca/mg ratio:4 : 1 (based on molarity)
- CaCl2 × 2H2O : 2.0 mmol/L (= 294.0 mg/L)
- MgSO4 × 7H2O : 0.5 mmol/L (= 123.0 mg/L)
- NaHCO3 : 0.75 mmol/L (= 65.0 mg/L)
- KCl : 0.075 mmol/L (= 5.8 mg/L)
Ratio of Ca : Mg = 4 : 1 (based on molarity)
Na : K = 10 : 1 (based on molarity)

OTHER TEST CONDITIONS
- Adjustment of pH: Yes,pH of the stock solution was adjusted to pH 8.0 ± 0.1 using 2 M NaOH
- pH : 7.8 to 8.1
- Photoperiod:16 h light : 8 h dark; 30 min dawn/dusk period was provided
- Light intensity:370 to 690 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Mortality and sublethal effects (at test start and after approximately 2, 24, 48, 72 and 96 hours),NOEC after 96 h, LOEC after 96 h; LC50: lethal concentration producing 50 % mortality after 96 h of exposure, LC0, LC 10 and LC100.

TEST CONCENTRATIONS
- Range finding study: NO, the lowest EC50 value of existing relevant algae or acute invertebrate (e.g. daphnia) tests was set as threshold concentration.
- Test concentrations: 100 mg/L
- Results used to determine the conditions for the definitive study:Since all EC50 values of existing relevant algae or acute invertebrate (e.g. daphnia) tests are above 100 mg test item/L, the only tested concentration was 100 mg test item/L.This threshold approach was performed in order to demonstrate that the test item had no toxic effects on the test fish up to 100 mg test item/L.
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
LC0
Effect conc.:
ca. 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
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
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
ca. 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
- Behavioural abnormalities: NO
- Observations on body length and weight:No
- Other biological observations: Dark colouration was observed in two fish after 24 h and in seven fish after 72 hour.
- Mortality of control: NO
- Other adverse effects control: It was observed after 96 hours that two fish were mainly on the bottom.
- Abnormal responses: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No remarkable observations were made concerning the appearance of the test media.

Table No.2: Mortality and Sublethal Effects

 

 

               Mortality and Sublethal Effects

          Nominal Concentration [mg test item/L]

 

 

 

Control

100

0 h

# mort

0

0

# symp

0

0

symp

-

-

2 h

# mort

0

0

# symp

0

0

symp

-

-

24 h

# mort

0

0

# symp

0

2

symp

-

DC

48 h

# mort

0

0

# symp

0

0

symp

-

-

72 h

 

 

# mort

0

0

# symp

0

7

symp

-

DC,MB

96 h

# mort

0

0

# symp

0

2

symp

-

MB

# mort: Number of dead fish

# symp: Number of fish with sublethal effects

AP: apathy

CO: convulsions

DA: distended abdomen

DC: dark colouration

EG: strongly extended gills

EX: exophthalmus

FF: fins clearly shortened or frayed out at the border

MB: fish mainly on the bottom

MS: mucous secretion

SB: fish lying on side or back on the bottom

SV: hyperventilation

TS: tumbling during swimming

WS: fish mainly at the water surface

Table No.3: Temperature [°C] in the Test Media

Nominal Concentration [mg test item/L]

                                       Water temperature [°C]

 

 

0h

24h

48h

72h

96h

Control

new

15

15

15

15

-

 

aged

-

15

15

15

15

100

new

14

15

15

15

-

 

aged

-

15

15

15

15

Table No.4: pH-Values in the Test Media

Nominal Concentration [mg test item/L]

                                       pH value

 

 

0h

24h

48h

72h

96h

Control

new

8.0

8.0

8.0

8.0

-

 

aged

-

7.9

7.9

8.0

8.0

100

new

8.0

7.9

8.1

8.0

-

 

aged

-

7.8

7.9

7.9

7.8

Table No.5: Dissolved Oxygen Concentrations [%] in the Test Media

Nominal Concentration [mg test item/L]

                                            O2[%]

 

 

0h

24h

48h

72h

96h

Control

new

100

99

98

100

-

 

aged

-

97

96

99

96

100

new

96

97

97

93

-

 

aged

-

99

98

98

99

Table No.6: Appearance of the Test Item in Test Medium

Nominal Concentration [mg test item/L]

                     Appearance of the Test Item in the Test Media

 

 

0h

24h

48h

72h

96h

100

new

0

0

0

0

-

 

aged

-

0

0

0

0

Abbreviations:

0: No remarkable observations, clear test medium

1: Colouration caused by the test item

2: Turbidity caused by the test item

3: Inhomogeneous dispersion of the test item

4: Precipitation of the test item

5: Test item at the surface

6: Test item lying at the bottom of the test vessel

7: Test item caused foaming

+: Effects were strongly pronounced

Effects in parenthesis were only slightly pronounced

Table 7. Results for the Determination of the Test Item in the Test Samples

Sample Description

[mg test item/L]

Age

 

 

Day  h

Concentration

     Found

[mg test item/L]1

D.F.

Concentration

     calculated

[mg test item/L]1

Concentration

     nominal

[mg test item/L]

% of

Nominal2

Control (diluted)

0

0

<LOD

2

n.a.

0.000

n.a.

Control (diluted)

0

0

<LOD

2

n.a.

0.000

n.a.

Control (diluted)

1

24

<LOD

2

n.a.

0.000

n.a.

Control (diluted)

1

24

<LOD

2

n.a.

0.000

n.a.

Control (diluted)

3

0

<LOD

2

n.a.

0.000

n.a.

Control (diluted)

3

0

<LOD

2

n.a.

0.000

n.a.

Control (diluted)

4

24

<LOD

2

n.a.

0.000

n.a.

Control (diluted)

4

24

<LOD

2

n.a.

0.000

n.a.

100

0

0

52.804

2

105.609

100.000

106

100

0

0

52.627

2

105.254

100.000

105

100

1

24

52.529

2

105.057

100.000

105

100

1

24

52.609

2

105.218

100.000

105

100

3

0

53.445

2

106.891

100.000

107

100

3

0

53.233

2

106.466

100.000

106

100

4

24

53.346

2

106.693

100.000

107

100

4

24

53.584

2

107.168

100.000

107

 

 

 

 

 

 

mean value (n=8):

106

 

 

 

 

 

 

RSD (n=8):

1

1 The tabulated results represent rounded results calculated on the exact raw data

2 The results represent rounded values

LOD: Limit of Detection = 1.1 mg test item/L

n.a.: not applicable; RSD: Relative Standard Deviation; D.F.: Dilution factor

Table 8. Results for the Determination of the Test Item in the Fortified Samples

Sample description

 

 [mg test item/L]

Concentration

     Found

[mg test item/L]1

D.F.

Concentration

     calculated

[mg test item/L]1

Concentration

     nominal

[mg test item/L]

% of

Nominal2

Analytical Blank Control

0

<LOD

2

n.a.

0.000

n.a.

Analytical Blank Control

0

<LOD

2

n.a.

0.000

n.a.

Fortified Sample

50

25.318

2

50.636

50.070

101

Fortified Sample

50

25.082

2

50.164

50.070

100

Fortified Sample

50

24.981

2

49.962

50.030

100

Fortified Sample

50

25.172

2

50.344

50.030

101

Fortified Sample

50

25.461

2

50.922

50.030

102

 

mean value (n=5):

101

RSD (n=5):

1

Fortified Sample

140

70.075

2

140.149

140.196

100

Fortified Sample

140

70.891

2

141.781

140.196140.084

101

Fortified Sample

140

70.051

2

140.102

140.084

100

Fortified Sample

140

68.981

2

137.961

140.084

98

Fortified Sample

140

68.853

2

137.706

 

98

 

mean value (n=5):

99

RSD (n=5):

1

Overal mean value (n=8):

100

RSD (n=8):

1

1 The tabulated results represent rounded results calculated on the exact raw data

2 The results represent rounded values

LOD: Limit of Detection = 1.1 mg test item/L

n.a.: not applicable; RSD: Relative Standard Deviation; D.F.: Dilution factor

Validity criteria fulfilled:
yes
Remarks:
The mortality in the controls must not exceed 10% (mortality=0% during the test), DOC <=60% of the air-saturation value throughout. (93 to 100 % of the air saturation value during the test)
Conclusions:
The test item does not cause the mortality at the concentrations 100mg/l. The LC50>100mg/l .
Executive summary:

In accordance with OECD guideline 203 and GLP study, the test item was tested to demostrate that it has no toxic effect on the test fish up to the threshold concentration of 100 mg test item/L. The single-concentration test (limit test) was performed. It was based on the derivation of a threshold concentration from relevant algae and acute invertebrate (e.g. daphnia) tests at which fish toxicity was tested. Two treatment group were used in this study (one test item concentration at nominal 100 mg/L and one control) each containing 7 individuals. The acute toxicity to unfed juvenile Rainbow Trout was determined in an aerated, semi static, 96-hour test. The tested fish were observed at test start and after approximately 2, 24, 48, 72 and 96 hours test duration for sublethal effects and mortality. The samples of the test medium were analysed via HPLC-method. Measured parameters in this study: 96-hour LC50: > 100 mg test item/L (nominal) 96-hour LC10: > 100 mg test item/L (nominal), 96-hour LC0: 100 mg test item/L (nominal), 96-hour LC100: > 100 mg test item/L (nominal), 96-hour NOEC: 100 mg test item/L (nominal), 96-hour LOEC: > 100 mg test item/L (nominal). It was observed after 96 hours that two fish were mainly on the bottom. The test item does not cause the mortality at the concentration 100 mg test item/L.

Description of key information

Key study: The test item does not cause the mortality at the concentrations 100 mg test item/L. LC50 (96h) >100 mg/L

Key value for chemical safety assessment

LC50 for freshwater fish:
100 mg/L

Additional information

Key study: In accordance with OECD guideline 203 and GLP study, the test item was tested to demostrate that it has no toxic effect on the test fish up to the threshold concentration of 100 mg test item/L. The single-concentration test (limit test) was performed. Two treatment group were used in this study (one test item concentration at nominal 100 mg/L and one control) each containing 7 individuals. The acute toxicity to unfed juvenile Rainbow Trout was determined in an aerated, semi static, 96-hour test. The tested fish were observed at test start and after approximately 2, 24, 48, 72 and 96 hours test duration for sublethal effects and mortality. The samples of the test medium were analysed via HPLC-method. In the control and the only test concentration of 100 mg test item/L, all fish survived until the end of the experiment. It was observed after 96 hours that two fish were mainly on the bottom. The result of the test: LC50 (96h) >100 mg/L