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Ecotoxicological information

Short-term toxicity to fish

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Administrative data

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From June 11, 2010 to June 20, 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study well documented, followed guideline, GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
according to OECD, EC and German principles of Good Laboratory Practice

Test material

Constituent 1
Chemical structure
Reference substance name:
2-methyl-p-phenylenediamine sulfate
EC Number:
210-431-8
EC Name:
2-methyl-p-phenylenediamine sulfate
Cas Number:
615-50-9
Molecular formula:
C7H10N2.H2O4S
IUPAC Name:
2-methylbenzene-1,4-diamine; sulfuric acid
Constituent 2
Reference substance name:
HAARBRAUN
IUPAC Name:
HAARBRAUN
Constituent 3
Reference substance name:
toluene-2,5-diamine sulfate
IUPAC Name:
toluene-2,5-diamine sulfate
Details on test material:
- Name of test material: Toluene-2,5-diamine sulfate; Haarbraun (TSIN # 23005)
- Substance type: Pure active substance 
- Physical state: White to light pink powder 
- Stability under test conditions: Not reported 
- Storage condition of test material: Room temperature, protected from moisture and light
- Solubility: 5.03 g/L

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0, 0.625, 1.25, 2.50, 5.00, 10.0 mg/L (all test concentrations)
- Sampling method: All test concentrations were sampled daily (until 100% mortality occurred). 
- Sample storage conditions before analysis: All samples were analysed directly after sampling.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION  
- Preparation: Stock solutions were prepared in HPLC water. For the test concentrations of 0.625 and 1.25 mg/L, stock solutions of 1.56 and 3.10 g/L were prepared. For the test concentrations of 2.5 to 10 mg/L, stock solutions of 5.00 g/L were prepared.
- Application of test solution: A flow-through exposure design was carried out. The stock solutions with different concentrations were introduced using precision syringe pumps. The dilution water was split in 6 separate streams. The stock solutions were mixed in a mixing chamber by magnetic stirring before passing the aquaria.

Test organisms

Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM 
- Common name: Zebrafish
- Strain: Not reported 
- Source: Niedersachsischer Landesbetrieb fur Wasserwirtschaft, Kusten- und Naturschutz, An der Scharlake 39, D-31135 Hildesheim. All fish used in the test were gained at Dr. U. Noack Laboratorien from a single brood stock. 
- Age at study initiation: Not reported
- Length at study initiation: 2.24 cm 
- Weight at study initiation: 0.180 g 
- Feeding during test: No

HOLDING CONDITIONS (Performed at test facility)
- Temperature: 23 ± 2°C 
- Light intensity: Diffuse light (0.1 - 10 µmol photons x m (2) x s (1), natural photoperiod). 
- Food: Dry food sera vipan SERA GMBH, D-52518 Heinsberg. The amount of food was 4% of the fish body weight per feeding day. 
- Feeding frequency: 3 times per week 
- Water (maintenance): Tap water of local origin was used for holding and testing. The water was filtered on activated charcoal and aerated for at least 24 h to remove chlorine. The water was analysed biannual according to German tap water regulation. Nominal water parameters were:
Total hardness: 10 - 250 mg CaCO3/L 
pH-value: 6.0 - 8.5 
Dissolved oxygen: >80% of the air saturation value
- The water was changed at least once per week. 
- Health: No disease treatments were administered throughout holding.

ACCLIMATION 
- Acclimation period: At least 12 d
- Acclimation conditions: The same water was used for holding and testing of fish. 
- Type, frequency and amount of food: See description of holding above.

Study design

Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
None

Test conditions

Hardness:
60.5 mg/L as CaCO3 (Measured by Spectrophotometer: DR 5000, HACH LANGE). The values were within the acceptable limits.
Test temperature:
22.8 - 23.5 C (Measured by Data Logger: 175-T3, TESTO (for continuous measuring)). The values were within the acceptable limits.
pH:
7.38-8.70 (Measured by HQ40d multi, HACH). The values were within the acceptable limits.
Dissolved oxygen:
100% (Measured by HQ40d multi, HACH) The values were within the acceptable limits.
Nominal and measured concentrations:
Nominal test concentrations: 0, 0.625, 1.25, 2.50, 5.00 and 10.0 mg/L 

Measured concentrations (geometric mean): 0, 0.553, 1.07, 2.21, 4.43 and 8.85 mg/L. The measured concentrations were 83 - 95% of the nominal concentrations throughout exposure, therefore the results were based on nominal concentrations.
Details on test conditions:
TEST SYSTEM 
- Test vessel: Glass-aquaria loosely covered by glass tops
- Type: Open 
- Material, size, headspace, fill volume: Each glass aquaria contained about 7 L of test medium. 
- Aeration: No (the test was a flow-through design)
- Type of flow-through: Membrane piston pumps provided the water flow-through. The dilution water was split into 6 separate streams. Precision syringe pumps introduced appropriate volumes of stock solutions with different concentrations. The stock solutions were mixed in a mixing chamber by magnetic stirring before passing the aquaria. 
- Equilibration period: An equilibration period of 5 d was carried out prior to exposure. The nominal test concentrations were analytically measured during the equilibration period. Details are provided in the study report (Table 10).
- Renewal rate of test solution: 20 times/d corresponding to flow rate of 5.28-5.76 L/h 
- No. of organisms per vessel: 10 
- No. of vessels per concentration: 1 
- No. of vessels per control: 1 
- Biomass loading rate: 1 fish/0.7 L
- Fish density in the vessel: < 1 g fish/L

TEST MEDIUM / WATER PARAMETERS 
- Source/preparation of dilution water: Same tap water was used for holding and testing. Detail of holding water is provided under ‘Details on test organisms’ section above.
- Intervals of water quality measurement: pH-value, temperature and oxygen saturation were measured daily in all vessels after start of exposure and every 24 h. Total hardness of the water was determined at test start. During the test the water temperature was recorded continuously in the control aquarium with a data logger. The flow rates of the test media were checked daily.

OTHER TEST CONDITIONS 
- Photoperiod: 12 h light/12 h dark 
- Light intensity: 0.01 - 0.04 µmol photons x m (-2) x s (-1). Dimmed light was used with regard to the test material properties.

EFFECT PARAMETERS MEASURED: Fishes were observed after 24, 48, 72 and 96 h for the following endpoints:  
1. Mortality (evident by no visible movement and if touching of the caudal peduncle produced no reaction)  
2. Visible abnormalities (e.g. loss of equilibrium, swimming behavior, respiratory function, pigmentation etc). 
3. Mean body weight of fish (at the end of exposure) 
4. Mean total length of fish (at the end of exposure)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: A 96 h non-GLP range finding test under flow-through conditions was carried out with five fish/concentration.  
- Test concentrations: 0, 0.1, 1.0 and 10.0 mg/L
- Results used to determine the conditions for the definitive study: Yes. Following results were observed in the range finding study: 
1. Mortality: 100% mortality was observed in 10.0 mg/L dose level within 24 h of exposure. No mortality was observed in any of the remaining test concentration throughout the range finding study. 
2. Behaviour: All the test organisms exposed with a concentration of 1.0, 0.1 and control exhibited normal behaviour throughout the range finding study.  

Details on range finding study is provided in the study report.
Reference substance (positive control):
not required

Results and discussion

Effect concentrations
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
1.08 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mortality (fish)
Remarks on result:
other: 95% CL: 0.799 - 1.44 mg/L
Details on results:
- Mortality in test groups: Refer to 'Table 2' under 'Any other information on results incl. tables' section. 
- Behavioural abnormalities: Slowed escape reflex, laying on the back, lethargy, no escape reflex, staggering, arresting on the ground were observed in fish at concentrations above 0.625 mg/L. Details on biological data at 24, 48, 72 and 96 h for different concentrations are provided in the study report (Table 5).
- Observations on body length and weight: Mean body weight: 0.178 g; Mean total length: 2.12 cm (ranged from 1.20 - 2.90 cm)
- Mortality of control: 0%
- Any observations that might cause a difference between measured and nominal values: No.
- Effect concentrations exceeding solubility of substance in test medium: No
Reported statistics and error estimates:
The LC50-values and corresponding 95% confidence intervals were calculated after 24, 48, 72 and 96 h from the nominal test material concentrations using probit analysis (EPA probit analysis program, version 1.5).

Any other information on results incl. tables

Sublethal observations / clinical signs:

Table 2: Cumulative Mortality (%) in the Test Vessels (Study # 66992)

Nominal concentration of Haarbraun (mg/L)

Test Duration (h)

24

48

72

96

10.0

100

100

100

100

5.00

100

100

100

100

2.50

70

100

100

100

1.25

10

40

60

60

0.625

0

10

10

10

Control

0

0

0

0

Study # 66992: Acute toxicity of Haarbraun (615 -50 -9) to Danio rerio.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The 96 hr LC50 of Toluene-2,5-diamine sulfate (Haarbraun) to Danio rerio (Zebrafish) was 1.08 mg/L. The result is based on nominal concentration (measured concentrations were 83-95% of nominal throughout the flow-through study).
Executive summary:

The 96 h acute toxicity study of Toluene-2,5-diamine sulfate (Haarbraun) to Danio rerio (Zebrafish) was determined under flow through conditions, following the OECD guideline 203 for fish acute toxicity test.

The nominal test concentrations were: 0, 0.625, 1.25, 2.50, 5.00 and 10.0 mg/L. Measured concentrations were 83 -95% of nominal throughout the test, and therefore results are based on nominal concentrations. Measured concentrations (geometric mean) were: 0, 0.553, 1.07, 2.21, 4.43 and 8.85 mg/L.

10 test organisms in single replicates were exposed to each test concentration and control. The dilution water was tap water (filtered, dechlorinated) of 60 mg/L hardness (as CaCO3).

The 96 h LC50 of Toluene-2,5-diamine sulfate (Haarbraun) to Danio rerio (Zebrafish) was 1.08 mg/L, based on nominal test concentration. 

This acute toxicity test is classified as acceptable and satisfies the guideline requirement for the OECD 203.