Reaction mass of (disodium 4-[[4-(acetylmethylamino)-2-sulphonatophenyl]amino]1-amino-9,10-dihydro-9,10-dioxoanthracene-2-sulphonate and sodium sulphate and sodium chloride)

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-05-25 to 2018-07-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Duplicate samples from the freshly prepared test medium of the only test concentration and the control were taken at the start of the test. For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentration during the test period, duplicate samples from the test medium of the only test concentration and the control were collected at the end of the test (after 96 hours) from the approximate centre of the aquaria.

Details on test solutions:

The test medium of the only test item concentration of nominal 158.7 mg test item/L was prepared by dissolving 1586.7 mg test item into 9998 mL test water by intense stirring for 10 minutes. The test media was prepared just before introduction of the test fish (= start of the test).

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

Species: Zebrafish (Danio rerio)
Size and Weight: Juveniles;
The mean body length of the fish* in the test was 2.12 cm ± 0.27 cm (Mean ± SD), the mean body wet weight 0.09 g ± 0.03g (Mean ± SD).
* 10 fish from the test fish batch were measured before the start of the test
Sex: Male and female
Origin: The test fish were obtained from ibacon in house breeding
Holding Conditions: All fish were obtained and held in the laboratory for at least 12 days before the start of the test. They were held in water of the quality to be used in the test for at least seven days immediately before testing under the following conditions:
Light: 16 hours photoperiod daily
Temperature: 21- 25 °C °C
Oxygen concentration: at least 80 % of the air saturation value
on one day a concentration of 75 % was measured
Feeding: three times per week or daily until 24 hours before the test was started
During the last 7 days prior to the start of the test no fish (0 %) died in the test fish batch. Therefore the mortalities in the fish batch were below 5 % and the fish batch was accepted.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

no

Hardness:

2.5 mmol/L (= 250.0 mg/L) as CaCO3

Test temperature:

22°C

pH:

7.7 to 7.9

Dissolved oxygen:

100 to 101 %

Nominal and measured concentrations:

158.7 mg test item/L corresponding to
100 mg active substance/L and a control
The lowest EC50 value of existing relevant Lemna gibba or acute invertebrate (e.g. daphnia) tests was set as threshold concentration. This threshold approach was performed in order to demonstrate that the test item has no toxic effects on the test fish up to the threshold concentration or at 100 mg (active substance)/L.
The quantification of the test item in the test samples was performed using liquid chromatography with UV detection. At the start of the test 91 % of the nominal test concentration was found. After 96 hours test duration, 93 % of the nominal value was determined. During the test the fish were exposed to a mean of 92 % of nominal.

Details on test conditions:

Test Environment: Controlled environmental room
Measurement of pH-Values, Dissolved Oxygen and Water Temperature:
The water temperature, pH-values and the dissolved oxygen concentrations were determined daily in the test media of each treatment group.
Water Temperature: 22°C
pH-Values of the Test Water: 7.7 to 7.9
Dissolved Oxygen Concentrations: 100 to 101 % of the air saturation value
Light Regime: 16 h light : 8 h dark; 30 min dawn/dusk period was provided
Light Intensity: 440 to 510 lux
Aeration of the Test Water: The test media were slightly aerated during the test.

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

158.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

> 158.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

158.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

In the control and the only test concentration of 158.7 mg test item/L, corresponding to 100 mg active substance/L, all fish survived until the end of the experiment and showed no sublethal effects during the exposure time.

Reported statistics and error estimates:

No statistical analysis was performed.
The LC50 could not be quantified due to the absence of toxicity of the test item. The LC0, NOEC and the LOEC were determined directly from the raw data.

Validity criteria fulfilled:

yes

Remarks:

In the control no fish died during the test. The dissolved oxygen concentration in the test media did not fall below 100 % of air saturation value during the test.

Conclusions:

The toxic effect of the test item to Zebrafish (Danio rerio) was assessed in a static limit test. Based on the test results the 96-hour LC0 was determined to be 158.7 mg test item/L based on nominal concentrations (corresponding to 100 a.s. mg/L). The NOEC was determined to be 158.7 mg test item/L (100 a.s. mg/L) and the LOEC was determined to be > 158.7 mg test item/L (100 a.s. mg/L) also based on nominal concentrations.

Executive summary:

The purpose of this study was to evaluate the acute toxicity of the test item to fish. For this purpose, juvenile Zebrafish were exposed in a static test to the only concentration under defined conditions for 96 hours. The recorded effects were the mortality and sublethal effects on the fish. A threshold approach was pursued.

The test method of application and the test system are recommended by the test guidelines and Zebrafish is one of the recommended test species.

This threshold approach was performed in order to demonstrate that the test item has no toxic effects on the test fish up to the threshold concentration or at 100 mg (active ingredient)/L. The lowest EC50 value of existing relevant algae or acute invertebrate (e.g. daphnia) tests was set as threshold concentration.

In the interest of animal welfare and efficient use of resources, it is important to avoid the unnecessary use of animals whenever possible. In the field of aquatic toxicology, this especially applies to the acute toxicity testing of fish according to OECD 203. The threshold approach described hereafter addresses fish toxicity by using a single-concentration test (limit test) requiring less fish compared to the full acute fish toxicity study. It is based on the derivation of a threshold concentration from relevant Lemna gibba and acute invertebrate (e.g. daphnia) tests at which fish toxicity is tested. If no mortality occurred in the limit test using the threshold concentration, the threshold concentration might be used as a surrogate LC50 value in the further hazard or risk assessment.

The purpose of the analytical part of this study was to verify the concentrations of the test item in the test medium.

Guidelines:

Commission Regulation (EC) No 440/2008, Annex, Part C, C.1.: "Acute Toxicity for Fish", Official Journal of the European Union, May 30, 2008

OECD Guideline for Testing of Chemicals, Section 2, No. 203, "Fish, Acute Toxicity Test", adopted July 17, 1992

OECD Series on Testing and Assessment, No. 23, "Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures", December 15, 2000

OECD Guidance Document, Series on Testing and Assessment No. 126, “Short Guidance in the Threshold Approach for Acute Fish Toxicity”, May 31, 2010

SANCO/3029/99 rev.4 11/07/00: Residues: Guidance for generating and reporting methods of analysis in support of pre-registration data requirements for Annex II (part A; Section 4) and Annex III (part A; Section 5) of directive 91/414

Materials and Methods

 

Test Species: Juvenile Zebrafish (Danio rerio) mean length: 2.12 ± 0.27 cm; source: ibacon breeding

Test Design: This study encompassed two treatment groups (one test item concentration at nominal 158.7 mg test item/L and one control) each containing 7 individuals. The acute toxicity to unfed juvenile Zebrafish was determined in an aerated, static, 96-hour test. The test fish were observed at test start and after approximately 2, 24, 48, 72 and 96 hours test duration for sublethal effects and mortality.

Endpoints: NOEC after 96 h, LOEC after 96 h; LC0: Highest test concentration at which no mortality occurred.

Test Concentration: 158.7 mg test item/L corresponding to 100 mg active substance/L and a control

Test Conditions: Water temperature: 22°C; pH value: 7.7 to 7.9; dissolved oxygen concentration: 100 to 101 % of the air saturation value; photoperiod: 16 h light - 8 h dark; light intensity: 440 to 510 lux and thus were within the ranges requested by guideline OECD 203.

Results:

Biological test results:

In the control and the only test concentration of 158.7 mg test item/L, all fish survived until the end of the experiment and showed no sublethal effects during the exposure time.

Analytical test results:

The quantification of the test item in the test samples was performed using liquid chromatography with UV detection. At the start of the test 91 % of the nominal test concentration was found. After 96 hours test duration, 93 % of the nominal value was determined. During the test the fish were exposed to a mean of 92 % of nominal.

Conclusion: The toxic effect of the test item to Zebrafish (Danio rerio) was assessed in a static limit test. Based on the test results the 96-hour LC0 was determined to be 158.7 mg test item/L based on nominal concentrations (corresponding to 100 a.s. mg/L). The NOEC was determined to be 158.7 mg test item/L (100 a.s. mg/L) and the LOEC was determined to be > 158.7 mg test item/L (100 a.s. mg/L) also based on nominal concentrations. The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part. All reported results refer to nominal values since the concentration of the test item were within ± 20 % of the nominal concentrations during the test.

This study is classified acceptable and satisfies the guideline requirements for fish acute test studies.

Description of key information

In the control and the only test concentration of 158.7 mg test item/L, all fish survived until the end of the experiment and showed no sublethal effects during the exposure time.