Formaldehyde, reaction products with m-phenylenediamine, sodium sulfide (Na2S) and sulfur

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:

03 September 2012 to 14 September 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. Conclusive valid guideline study under GLP conditions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), C1.

Deviations:

no

Principles of method if other than guideline:

A limit test was performed in accordance with the threshold approach requiring that the fish test is performed with a single concentration close to or slightly higher than the lowest EC50 value obtained in tests with in this case lemna and daphnia. The threshold approach (based on the Guidance on Information Requirements and Chemical Safety Assessment, ECHA (2008)) was used to demonstrate that zebra fish is not the most sensitive species for C.I. Leuco Sulphur Brown 37.

GLP compliance:

yes (incl. QA statement)

Remarks:

swissmedic, decision: 19-November-2010

Analytical monitoring:

no

Details on sampling:

On request of the Sponsor no analytical work was performed.

Vehicle:

no

Details on test solutions:

The preparation of the test medium was performed as far as possible in the dark to avoid photolytic degradation of the test item.

Due to the low water solubility of the test item, a dispersion with the loading rate of 100 mg/L was prepared at the start of the test and before each test medium renewal by dispersing 300 mg of the test item (dosing range: 300.0-300.5 mg) in 3 L of test water.

This preparation was supported by ultrasonic treatment for 15 minutes and intense stirring on a magnetic stirrer over 96 hours at room temperature in the dark, to dissolve a maximum amount of the test item in the dispersion. No auxiliary solvent or emulsifier was used. The pH of the test medium was 7.2.

After the 96-hour stirring period, the dispersion of the test item was filtered through a membrane filter (Schleicher & Schuell, Type NC20, pore size 0.2 µm).

The undiluted filtrate was used as stock solution and diluted with the test water to prepare the test medium of the single theoretical test concentration of 50 mg/L. The test medium was prepared just before the introduction of the fish (= start of the exposure) and prior to each test medium renewal.

The preparation of the test medium is based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

The study was performed with zebra fish (Brachydanio rerio). The fish were obtained from a breeding culture at Harlan Laboratories Ltd.. No medication was applied during holding and acclimatization. Prior to test start, the test fish were acclimatized for one week to the test water and temperature. During holding and acclimatization until one day before the start of the test, the fish were fed with a commercial fish diet (Tetra Min Hauptfutter, supplied by TETRA-Werke, 49304 Melle / Germany). During the last two weeks prior to the test, no fish died in the test fish batch and all fish were healthy.

From the acclimatized test fish batch, 10 fish were measured at the start of the test. The mean body length of the fish was 3.0 ± 0.2 cm, the mean body wet weight was 0.21 ± 0.03 g. These measured fish were not introduced for the test.

The test method and test species are recommended by the international test guidelines.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

not applicable

Hardness:

The Water Hardness was 1.25 mmol/L.

Test temperature:

The water temperature was 21 °C during the test (see attached Table 4).

pH:

The pH value in all treatments was 7.2 (see attached Table 2).

Dissolved oxygen:

The oxygen concentration was at least 8.5 mg/L (see attached Table 3), corresponding to at least 95% oxygen saturation.

Salinity:

According to OECD test guideline.

Nominal and measured concentrations:

Due to the low water solubility of the test item, a dispersion of the test item with the loading rate of 100 mg/L was ultrasonic treated for 15 minutes and continuously stirred at room temperature in the dark over 96 hours. Then, the dispersion was filtered. The dilution 1:2 of the undiluted filtrate with the loading rate of 100 mg/L was used as test medium. This corresponds to a theoretical concentration of 50 mg/L. Additionally, a control was tested in parallel.

Details on test conditions:

Reconstituted test water was used in the study. For further details on the Test Water please see section "Any other information on materials and methods incl. tables" below.

One glass test vessel with 5 liters of test medium was used for each treatment. The test vessels were labeled with the study number and all necessary additional information to ensure unique identification.

The water temperature in the test vessels was maintained at 21 °C. The test vessels were slightly aerated during the test period.

Since the test item is not stable under light conditions, the test was performed as far as possible in the dark to avoid photolytic degradation of the test item. The aquaria were wrapped with black plastic foil.

The test duration was 96 hours and the fish were not fed during the test.

A limit test was performed in accordance with the threshold approach requiring that the fish test be performed with a single concentration close to or slightly higher than the lowest EC50 value obtained in tests with algae and daphnia. The threshold approach (based on the Guidance on Information Requirements and Chemical Safety Assessment, ECHA (2008)) was used to demonstrate, that zebra fish is not the most sensitive species for C.I. Leuco Sulphur Brown 37.
A semi-static test with test medium renewal after 24 hours was performed to keep the concentrations of the test item in the test media as constant as possible during the test period of 96 hours.

During this semi-static test, the test fish were daily placed into a new test vessel with freshly prepared test medium of the corresponding treatment.

At the start of the exposure, 7 fish were introduced into each test vessel in a random order. The loading rate was 0.29 g fish wet weight per liter test medium. Thus, the requirement of a loading rate not exceeding 1 g fish/L was fulfilled.

Reference substance (positive control):

no

Remarks:

according to animal welfare reasons.

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

>= 50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

>= 50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

> 50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

> 50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

The biological results based on the theoretical concentration 50 mg/L are listed in the attached Table 1.The calculations are based on this theoretical concentration as no analytical determination could be conducted.

In the control and the test medium with the theoretical concentration of 50 mg/L (dilution 1:2) all fish survived until the end of the test and no visible abnormalities were observed at the test fish. Therefore, the 96 hour NOEC and LC0 of C.I. Leuco Sulphur Brown 37 to zebra fish were both determined to be at least 50 mg/L. The 96 hour NOEC and LC0 might even be higher than this concentration, but concentrations in excess of 50 mg/L have not been tested (threshold approach). The 96 hour LOEC, LC50 and LC100 were clearly higher than 50 mg/L (dilution 1:2). These values could not be quantified due to the absence of toxicity of C.I. Leuco Sulphur Brown 37 at the tested nominal concentration.

No remarkable observations were made concerning the appearance of the test medium. It was a clear solution throughout the entire test duration.

The pH value in all treatments was 7.2 (see attached Table 2). The oxygen concentration was at least 8.5 mg/L (see attached Table 3), corresponding to at least 95% oxygen saturation. The water temperature was 21 °C during the test (see attached Table 4).

The test was considered to be valid, since no mortality in the control was observed and the validity criterion of at least 60% oxygen saturation was fulfilled.

Results with reference substance (positive control):

not applicable according to animal welfare reasons.

Reported statistics and error estimates:

The NOEC and LC0 were determined directly from the raw data. The LOEC, LC100 and LC50 at the observation times could not be quantified due to the absence of a toxic effect of the test item at the tested nominal concentration.

Sublethal observations / clinical signs:

The biological test results (based on theoretical concentrations) after 96 hours exposure:

 

– 96-hour LC50:	>50 mg/L
– 96-hour LC0:	>=50 mg/L
– 96-hour LC100:	>50 mg/L
– 96-hour NOEC:	>=50 mg/L
– 96-hour LOEC:	>50 mg/L

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, the test item C.I. Leuco Sulphur Brown 37 had no acute toxic effects on zebra fish under the present test conditions at the theoretical concentration of 50 mg/L. This concentration corresponds to the lowest EC50 value obtained in the test with daphnia. According to the threshold approach it was demonstrated, that zebra fish is not the most sensitive species for C.I. Leuco Sulphur Brown 37.

Executive summary:

The acute toxicity of the test item C.I. Leuco Sulphur Brown 37 to zebra fish (Brachydanio rerio) was determined in a 96‑hour semi-static test with daily test medium renewal according to the EU Commission Directive 92/69/, Part C.1, the Commission Regulation (EC) No 440/2008, Part C.1 and the OECD Guideline for Testing of Chemicals No. 203 (1992).

A limit test was performed in accordance with the threshold approach requiring that the fish test is performed with a single concentration close to or slightly higher than the lowest EC₅₀value obtained in tests with in this case lemna and daphnia. The threshold approach (based on the Guidance on Information Requirements and Chemical Safety Assessment, ECHA (2008)) was used to demonstrate that zebra fish is not the most sensitive species for C.I. Leuco Sulphur Brown 37.

 

TheEC₅₀value for C.I. Leuco Sulphur Brown 37 in a lemna 7-day growth inhibition test was >100 mg/L (based on theoretical concentrations, Harlan Study No. D55221). The lowest EC₅₀value for C.I. Leuco Sulphur Brown 37 was obtained in a daphnia 48-hour immobilization test (Harlan Study No. D55210) and was 48 mg/L (based on theoretical concentrations). Based on this, the theoretical concentration tested in the present study with zebra fish was 50 mg C.I. Leuco Sulphur Brown 37/L (dilution 1:2).

 

Due to the low water solubility of the test item, a dispersion of the test item with the loading rate of 100 mg/L was ultrasonic treated for 15 minutes and continuously stirred at room temperature in the dark over 96 hours. Then, the dispersion was filtered. The dilution 1:2 of the undiluted filtrate with the loading rate of 100 mg/L was used as test medium. This corresponds to a theoretical concentration of 50 mg/L. Additionally, a control was tested in parallel.

 

The preparation of the test media was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (2000).

 

The biological test results (based on theoretical concentrations) after 96 hours exposure:

 

– 96-hour LC50:	>50 mg/L
– 96-hour LC0:	>=50 mg/L
– 96-hour LC100:	>50 mg/L
– 96-hour NOEC:	>=50 mg/L
– 96-hour LOEC:	>50 mg/L

 

In conclusion, the test item C.I. Leuco Sulphur Brown 37 had no acute toxic effects on zebra fish under the present test conditions at the theoretical concentration of 50 mg/L. This concentration corresponds to the lowest EC₅₀value obtained in the test with daphnia. According to the threshold approach it was demonstrated, that zebra fish is not the most sensitive species for C.I. Leuco Sulphur Brown 37.

Description of key information

In conclusion, the test item C.I. Leuco Sulphur Brown 37 had no acute toxic effects on zebra fish under the present test conditions at the theoretical concentration of 50 mg/L. This concentration corresponds to the lowest EC50 value obtained in the test with daphnia. According to the threshold approach it was demonstrated, that zebra fish is not the most sensitive species for C.I. Leuco Sulphur Brown 37.

 

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

EC50

Effect concentration:

50 mg/L

Additional information

 

The acute toxicity of the test item C.I. Leuco Sulphur Brown 37 to zebra fish (Brachydanio rerio) was determined in a 96‑hour semi-static test with daily test medium renewal according to the EU Commission Directive 92/69/, Part C.1, the Commission Regulation (EC) No 440/2008, Part C.1 and the OECD Guideline for Testing of Chemicals No. 203 (1992).

 

A limit test was performed in accordance with the threshold approach requiring that the fish test is performed with a single concentration close to or slightly higher than the lowest EC₅₀value obtained in tests with in this case lemna and daphnia. The threshold approach (based on the Guidance on Information Requirements and Chemical Safety Assessment, ECHA (2008)) was used to demonstrate that zebra fish is not the most sensitive species forC.I. Leuco Sulphur Brown 37.

 

TheEC₅₀value for C.I. Leuco Sulphur Brown 37 in a lemna 7-day growth inhibition test was >100 mg/L (based on theoretical concentrations, Harlan Study No. D55221). The lowest EC₅₀value for C.I. Leuco Sulphur Brown 37 was obtained in a daphnia 48-hour immobilization test (Harlan Study No. D55210) and was 48 mg/L (based on theoretical concentrations). Basedon this, the theoretical concentration tested in the present study with zebra fish was50 mgC.I. Leuco Sulphur Brown 37/L (dilution 1:2).

 

Due to the low water solubility of the test item, a dispersion of the test item with the loading rate of 100 mg/L was ultrasonic treated for 15 minutes and continuously stirred at room temperature in the dark over 96 hours. Then, the dispersion was filtered. The dilution 1:2 of the undiluted filtrate with the loading rate of 100 mg/L was used as test medium. This corresponds to a theoretical concentration of 50 mg/L. Additionally, a control was tested in parallel.

 

The preparation of the test media was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (2000).

 

The biological test results (based on theoretical concentrations) after 96 hours exposure:

 

– 96-hour LC50:	>50 mg/L
– 96-hour LC0:	>=50 mg/L
– 96-hour LC100:	>50 mg/L
– 96-hour NOEC:	>=50 mg/L
– 96-hour LOEC:	>50 mg/L

 

In conclusion, the test item C.I. Leuco Sulphur Brown 37 had no acute toxic effects on zebra fish under the present test conditions at the theoretical concentration of 50 mg/L. This concentration corresponds to the lowest EC₅₀-value obtained in the test with daphnia. According to the threshold approach it was demonstrated, that zebra fish is not the most sensitive species for C.I. Leuco Sulphur Brown 37.