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Short-term toxicity to fish

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Endpoint:
short-term toxicity to fish
Type of information:
other: experimental study on similar substance
Adequacy of study:
key study
Study period:
17 May 2016 to 31 May 2016
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
1992
Qualifier:
according to guideline
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Version / remarks:
2008
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Remarks:
No analytical method is available for the multi-constituent test substance. See 'Details on analytical methods' for further information.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test treatments were prepared separately for each test vessel. The test substance was weighed into a beaker. The defined volume of test water was filled into the test vessel. An aliquot from the test water in the test vessel was taken and mixed with the test substance using a high shear mixer (Ultra Turrax, IKA Werke GmbH &Co. KG, Germany) and the mixture was poured into the test vessel and the whole test batch was homogenized again with an ultra turrax. The test treatments were then stirred for about 2 days. Undissolved test substance was removed by filtration with a paper filter. The test solutions were filtered again with membrane filter (Whatman, pore width 0.2 μm, OE66). The first 50-100 mL of filtered solution was discarded (used to condition the filter). After filtering the solution was stirred for 10 min to ensure adequate dissolved oxygen levels. The control group was treated in the same way.
- Preparation scheme: see 'Any other information on materials and methods incl. tables'.
- Evidence of undissolved material: The control test treatment was clear. The 100 mg/L test treatment appeared clear and violet for up to 72h. At 72h it was turbid dark violet. After 96 hours the test treatment appeared turbid, dark violet and test substance was visible at the bottom of the test vessel.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebrafish
- Source: The fish were hatched at the testing facility.
- Body length: 3.9 cm (range 3.5 cm – 4.2 cm); Measurements were performed with all surviving fish from the control and lowest test concentration tanks at the end of exposure as a representative sample of the fish batch.
- Body weight: 0.48 g (range 0.30 g – 0.59 g); Measurements were performed with all surviving fish from the control and lowest test concentration tanks at the end of exposure as a representative sample of the fish batch.
- Age of the animals: Approx. 9 months
- Hatching date: 30 Aug - 01 Sept 2015

ACCLIMATION
- Acclimation period: at least 14 days
- Acclimation conditions: same as test
- Housing: Prior to testing, the batch of fish are housed in a glass tank (approx.45 L) receiving a continuous supply of fresh test water.
- Photoperiod: 16 hours light, 8 hours dark
- Water quality: The water used for housing and test purposes is nonchlorinated charcoal filtered drinking water (Frankenthal, Germany) mixed with deionized water and aerated to achieve the following water characteristics. The mixed test water is sanitized by UV treatment prior to entering the aquaria. Total hardness: Approx. 1 mmol/L = 100 mg/L CaCO3; Conductivity: Approx. 250 μS/cm (at 25°C); Ca content: Approx. 40 mg/L; Mg content: Approx. 5 mg/L; Acid capacity: Approx. 2.5 mmol/L; pH-value: Generally 7.5 – 8.5; Oxygen content: >80% saturation; TOC: Generally <2mg/L
- Temperature: 23 ± 1°C
- Diet: Commercial fish diet ”Tetramin“ (manufacturer Tetra-Werke, Melle, Germany) daily ad libitum, additionally generally on workdays live juvenile brine shrimp (Artemia nauplii).
- Withdrawal of feed: No feeding from approximately 24 hours before start of exposure
- Medical treatment: None during acclimatization
- Mortality during the last week before start of exposure: 0%
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Hardness:
100 mg/L CaCO3
Test temperature:
22 - 23 °C
pH:
8.3 - 8.6
Dissolved oxygen:
7.8 - 8.3 mg/L
Conductivity:
250 μS/cm
Nominal and measured concentrations:
- Nominal loading rate: 0 (control) and 100 mg test substance/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Approx. 10 Liter stainless steel aquaria (29x21x22 cm)
- Type (delete if not applicable): open / closed
- Test volume: 10 L
- Aeration: Continuous slight aeration via glass capillary.
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 7 Fish/ test vessel, 0.34 g fish/L
- Randomization: The test organisms were introduced into the test vessels according to a randomization plan prepared by using a program of the laboratory data evaluation group of the testing facility.
- Feeding: none

TEST MEDIUM / WATER PARAMETERS
- Water: similar to the water used during acclimation period, see 'Details on test organisms'.
- Intervals of water quality measurement: The appearance of the test solutions and dissolution behavior of the test substance was recorded daily. Temperature, oxygen content and pH-value were measured in each test vessel within 1 hour after start of exposure and after 24, 48, 72 and 96 hours. Additionally the TOC (Total organic carbon) values of the test concentration and the control was analyzed after start and at the end of the exposure.

OTHER TEST CONDITIONS
- Adjustment of pH: No adjustment of the pH-value
- Photoperiod: 16h light, 8h dark
- Light intensity: 114 - 431 Lux (the light intensity is determined in regular intervals at the surface of the aquaria)

EFFECT PARAMETERS MEASURED
- Mortality and toxic signs: the fish were observed for mortality and toxic signs (changes appearance and abnormal behavior in comparison to the control group) within 1 hour after start of exposure and 6, 24, 48, 72 and 96 hours after start of exposure. Fish were considered dead if there was no visible movement and no reaction after touching.
- Body weight: At the end of exposure the body wet weight and total length of a representative sample of fish were determined. The representative sample consists of all surviving fish from the lowest concentration and control group.

TEST CONCENTRATIONS
- Results used to determine the conditions for the definitive study: In the first experiment mortality after 24 hours was observed, which may be due to physical effects, because undissolved test substance was not removed.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate
Basis for effect:
mortality (fish)
Details on results:
MORTALITY
No deaths were observed throughout the test.

GENERAL SYMPTOMS OF TOXICITY
All fish displayed normal behavior. No other visual signs of toxicity were observed.

OBSERVATIONS ON TEST SOLUTIONS
The control test treatment was clear. The 100 mg/L test treatment appeared clear and violet for up to 72h. At 72h it was turbid dark violet. After 96 hours the test treatment appeared turbid, dark violet and test substance was visible at the bottom of the test vessel.

OTHER FINDINGS
- Water quality: TOC concentrations in the control at the start and end of the experiment were 2.4 mg/L and 1.0 mg/L, respectively. TOC concentrations in the 100 mg/L test substance loading rate at the start and end of the experiment were 16.0 mg/L and 13.8 mg/L, respectively. The higher TOC in the 100 mg/L treatment demonstrates that the test organisms were exposed to soluble organic components from the test substance.
Reported statistics and error estimates:
Since the study is designed as a limit test, the data were not appropriate for the use of computerized methods for the calculation of point estimates. Any reported point estimates (NOEC and LCx values) are estimated based on expert judgment in accordance with test guideline recommendations. The test endpoints were calculated and reported based on the nominal test substance loading rate.

Validity criteria fulfilled:
yes
Conclusions:
Not toxic to fish
LC 50 > 100 mg/l
Endpoint:
short-term toxicity to fish
Type of information:
other: experimental study on similar substance
Adequacy of study:
supporting study
Study period:
7 Nov 1988 to 11 Nov 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods
Qualifier:
equivalent or similar to guideline
Guideline:
other: DIN 38 412 “ Testverfahren mit Wasserorganismen (Gruppe L). Allgemeine Hinweise zur Planung, Durchführung und Auswertung biologischer Testverfahren" und "Bestimmung der Wirkung von Wasserinhaltsstoffen auf Fische - Fischtest (L15)", June 1982
Version / remarks:
June, 1982
GLP compliance:
no
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: In the form of an aqueous emulsion (10 g/L) the product was added to the test water; subsequently the fish were placed into the aquaria.
Test organisms (species):
Leuciscus idus
Details on test organisms:
TEST ORGANISM
- Common name: Golden Orfe
- Strain: golden variety
- Source: Fischzucht Paul Eggers, D-2354 Hohenwestedt, FRG
- Body length: 7.0 cm (range: 6.0 - 8.5)
- Body weight: 4.4 g (range: 2.8 - 7.1)
- Corpulence factor of the batch: 1.3

HOUSING
- The fish were kept in a flow-through tank in tap water cleaned by active carbon and aerated with oil-free air.
- Photoperiod: 16 hours light and 8 hours dark
- Total hardness: about 2.5 mmol/L
- Acid capacity: about 5.5 mmol/L
- Oxygen content: >60% of maximum saturation
- pH: about 8.0
- Duration of housing and adaptation: about 2 months
- Adaptation to test water and test temperture: 3 days
- Water temperature: 19 - 22 °C during housing, 20°C during adaptation period and test
- Mortality during the last 2 weeks of housing: about 0.07%
- Mortality during the adaptation period: 0%
- Medical treatment: twice with 0.05 mg/L malachite green chloride; once with 10 mg/L tetracycline hydrochloride
- Diet: ad libitum growing feed F/B 50, SSNIFF Spezialdiaeten GmbH, D-4770 Soest, FRG
- Withdrawel of food before exposure: 1 day
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
2.5 mmol/L
Test temperature:
20 °C
pH:
7.4 - 7.6
Dissolved oxygen:
8.1 - 8.9 mg/L
Nominal and measured concentrations:
- Nominal test item concentrations: 0 (control), 46.4, 100, 215 and 464 mg/L
Details on test conditions:
TEST SYSTEM
- Aeration: slight continuous aeration with oil-free air
- Volume of water: 10 liters
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Loading (g fish/ L test water): 4.4
- Test vessel: all-glass aquarium (30 cm x 22 cm x 24 cm)

TEST MEDIUM
- Source/preparation of dilution water: Reconstituted freshwater according to DIN 38 412, part 11,October 1982, preparation from fully demineralized tap water (conductivity max. 10 μMHO), resalting is carried out by addition of 294.0 mg/L CaCl2·2H2O, 123.3 mg/L MgSO4·7H2O, 63.0 mg/L NaHCO3 and 5.5 mg/L KCl.

WATER PARAMETERS OF TEST WATER READY FOR USE
- Total hardness: 2.5 mmol/L
- Acid capacity: 0.8 mmol/L
- Ratio Ca/Mg ions: 4 : 1
- Ratio Na/K ions: 10 : 1
- pH: about 8.0

OTHER TEST CONDITIONS
- Test temperature: 20 °C
- Photoperiod: 16 hours light and 8 hours dark

EFFECT PARAMETERS MEASURED:
- Survival and symptoms (at t = 1, 4, 24, 48, 72 and 96 h)

TEST CONCENTRATIONS
- Reason for selection of the concentrations: based on the results of range finding studies and main studies not evaluated (LC50 after 96 h: between 100 and 320 mg/L)
Reference substance (positive control):
yes
Remarks:
Chloroacetamide
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
ca. 220 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Details on results:
GENERAL OBSERVATION
The aquarium contents were intensely coloured and cloudy.

MORTALITY
No mortality was observed in the control, 46.4 and 100 mg/L test concentration. At the test concentration of 215 mg/L 2 fish (20%) were dead after 24 hours exposure and 4 fish (40%) were dead after 48 hours. At the highest tested concentration all fish had died after 24 hours exposure. See 'Any other information on results incl. tables'.

SYMPTOMS:
- Behavioural abnormalities: Apathy and narcotic-like state was observed at 215 mg/L after 24 hours; narcotic-like state was observed at 464 mg/L after 4 hours. Since the test solutions were intensely coloured and cloudy a complete evaluation of the symptoms could not be performed.
Results with reference substance (positive control):
Chloroacetamide LC50 after 48 h: about 31 mg/L (determined 10 Oct. 1988), this lethal concentration corresponds to the normal sensitivity. This respective test was valid.
Reported statistics and error estimates:
Determination or calculation of the median lethal concentration (LC 50) and, if possible the LC 5 and the LC 95 using the probit analysis.
Sublethal observations / clinical signs:

Table: Results on fish survival and symptoms

Nominal conc.

(mg/L)

Number of fish

 

Dead* fish after

 

1h

4h

24h

48h

72h

96h

46.4

10

0

0

0

0

0

0

100

10

0

0

0

0

0

0

215

10

0

0

2

4

4

4

464

10

0

0

10

10

10

10

 

 

 

 

 

 

 

 

0

10

0

0

0

0

0

0

 

 

 

 

 

 

 

 

Nominal conc.

(mg/L)

 

 

Symptoms*

 

1h

4h

24h

48h

72h

96h

46.4

 

 

 

 

 

 

 

100

 

 

 

 

 

 

 

215

 

 

 

an

 

 

 

464

 

 

n

 

 

 

 

 

 

 

 

 

 

 

 

0

 

 

 

 

 

 

 

Explanation of symptoms:

a=apathy, b=abdominal distension, e=exophthalmos,f=escapereflex, h=hyperreflexia,k=convulsions,l=gasping, n=narcotic-likestate,t=tumbling,u=restlessness,v=discoloration,w=headstand,x=accelerated respiration

 

* for checking for dead fish and for possible symptoms the fish were directed towards the front pane of the aquaria using a net.

 

Table: Effect values

LC50 (mg/L; nominal concentrations) after

1h

greater

460 (mg/L)

(1% significance level)

4h

greater

460 (mg/L)

(1% significance level)

24h

greater

220 (mg/L)

(10% significance level)

 

lower

460 (mg/L)

(1% significance level)

48h

about

220 (mg/L)

 

72h

about

220 (mg/L)

 

96h

about

220 (mg/L)

 

 

Symptoms: Since the test solutions were intensely coloured and cloudy a complete evaluation of the symptoms could not be performed.

                       

No Observable Effect Concentration: 100 mg/l

Maximum concentration causing no mortality: 100 mg/l

Minimum concentration causing 100% mortality: 464 mg/l

Validity criteria fulfilled:
yes
Conclusions:
Not toxic to fish
LC50 = 220 mg/l

Description of key information

The 96-h LL50 value is >100 mg/L (based on nominal test substance loading rate) in freshwater fish (Danio rerio).

Key value for chemical safety assessment

Additional information

The short-term toxicity of the analogue substance to freshwater fish was determined in two studies performed on similar substance; one with the dried substance and one with the liquid (non-dried) substance.

In the key study, the short-term toxicity of the dried substance to freshwater fish was determined in a limit test according to OECD TG 203 and in compliance with GLP criteria (BASF, 2017). In this study, groups of 7 zebrafish (Danio rerio) were exposed to nominal loading rates of 0 (control) and 100 mg test substance/L for 96 hours under static conditions. Test concentrations were not analytically verified because a reliable method for analysis in the required concentration range could not be developed. Since all reasonable efforts were taken to produce a WAF of the test substance in the test media following the guidance of OECD 23, results are based on the loading rate. The appearance of the test solutions and dissolution behavior of the test substance was recorded daily. Fish mortality and general symptoms of toxicity were recorded within 1 hour after start of exposure and after 6, 24, 48, 72 and 96 hours exposure. In this study the OECD validity criteria are met. The 100 mg/L test treatment appeared clear and violet for up to 72h. At 72h it was turbid dark violet. After 96 hours the test treatment appeared turbid, dark violet and test substance was visible at the bottom of the test vessel. No mortality or visual signs of toxicity were observed during the test. Based on these findings the 96-h LL50 value was determined at >100 mg/L.

Based on the read across considerations same results apply to Acid Black 63.

This result is supported by data from a second study, where the short-term toxicity of the liquid substance to freshwater fish was determined according to DIN 38 412 (BASF, 1989). In this study, groups of 10 Golden Orfe (L. idus) were exposed to nominal test substance concentrations of 0 (control), 46.4, 100, 215 and 464 mg/L for 96 hours under static conditions. Test concentrations were not analytically verified. Incidences of mortality and clinical effects were recorded after 1, 4, 24, 48, 72 and 96 hours exposure. Since test solutions were intensely coloured and cloudy a complete evaluation of the symptoms could not be performed. Apathy and narcotic-like state was observed at 215 mg/L after 24 hours; narcotic-like state was observed at 464 mg/L after 4 hours. No mortality was observed in the control, 46.4 and 100 mg/L test concentration. At the test concentration of 215 mg/L 2 fish (20%) were dead after 24 hours exposure and 4 fish (40%) were dead after 48 hours. At the highest tested concentration all fish had died after 24 hours exposure. Based on these findings the 96-h LC50 is determined at ca. 220 mg/L. Even though non-dissolved test substance was not removed from the medium, meaning the substance could e.g. possibly clog or goat gills, this exposure was considered "worst-case", allowing the results to be used as supporting information.