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Diss Factsheets

Ecotoxicological information

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From Nov. 14, 2012 to Dec. 5, 2012
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:
2012

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
GLP compliance:
yes
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:
toluene-2,5-diamine sulfate
IUPAC Name:
toluene-2,5-diamine sulfate
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material: Toluene-2,5-diamine sulfate (Code: 95353654)
- TSIN: WR23005
- Substance type: Pure active substance
- Physical state: White to light pink or light grey powder
- Stability under test conditions: Not reported
- Storage condition of test material: Room temperature, protected from moisture and light
- Water solubility: 5.03 g/L

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0, 0.160, 0.250, 0.400, 0.630, 1.00 mg/L
- Sampling period: Samples were collected on Days 0, 1, 7, 8, 14, 15 (fresh media, 0 hours) and on Days 1, 2, 8, 9, 15, 16 (old media, 24 hours) of all concentration levels and the control
- Sampling method: Directly after preparation of the test solutions, the fresh media samples were taken in duplicate. Sampling was done in brown glass vials. For each sample, 0.8 mL of the test solution were diluted for stabilization with 3.2 mL HPLC water (containing 20 % acetonitrile, 0.2 % TFA and 20 % of a 0.2 % ascorbic acid solution) and analysed the day of collection within 4 hours of sampling.
For the old media, samples were taken directly from the test vessels containing daphnids. The old media samples were prepared as described for the fresh media samples. All samples were analysed the day of collection within 4 hours of sampling.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION OF TEST SOLUTION: The stock solution (1.00 mg/L test material was weighed out) was daily prepared with dilution water and immediately used for preparation of the test solution. The stock solution was stirred at approximately 1100 rpm for 5 min at room temperature. The test concentrations were prepared by diluting the stock solution with dilution water.
- Controls: Dilution water without test material.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Strain/clone: Clone 5
- Source: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu)
- Age at study initiation: 2 to 24 hours old
- Culturing of daphnids: All daphnids were cultured in a glass vessel (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20  2 °C, 16 h illumination (light intensity of max. 20 µEm (-2)  s(-1). The culture medium was Elendt M4, according to Elendt (1990), modified to a total hardness of 160 to 180 mg CaCO3/L. Daphnid cultures were fed at least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus, with an algae cell density of > 10(6) cells/mL
- Feeding during test: Yes
- Food type and amount: Pseudokirchneriella subcapitata (0.157 – 0.275 mL) and Desmodesmus subspicatus (0.190 - 0.270 mL) suspension was provided as food corresponding to 0.2 mg C per Daphnia and day. Variation was according to the density of the algae suspension, but was the same for all test groups on each feeding day
- Frequency: Daily

ACCLIMATION
- Acclimation period: Not reported. However, daphnids were cultured under the same conditions as the test
- Acclimation conditions: Same as test
- Type and amount of food: See description of culture above.
- Feeding frequency: See description of culture above.

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Post exposure observation period:
None

Test conditions

Hardness:
162 to 180 mg CaCO3/L
Test temperature:
The water temperature in the test vessels was within the range 19.9 to 21.4°C. The incubator temperature was in the range of 18 to 20°C.
pH:
Overall pH range: 7.74 -8.63
Nominal and measured concentrations:
Nominal test concentrations: 0, 0.160, 0.250, 0.400, 0.630, 1.00 mg/L
Time weighed mean measured test concentrations: 0.0620, 0.107, 0.175, 0.276, 0.364 mg/L. The measured concentrations of the test item were in the range of 94 to 110 % of the nominal values in the fresh media (0 hours) and < LOQM to 41 % in the old media (24 hours).
All results were based on both nominal and time weighed mean measured test concentrations of test material.
Details on test conditions:
TEST SYSTEM
- Test vessel: Flasks
- Type: Closed (with screw caps)
- Material, fill volume: Sealed glass flasks (4.5 (ID) x 9.5 (H) cm) with screw caps (made from polypropylene) were used. They were filled up with the test solutions (ca. 130 mL) having nearly no headspace to reduce contact with oxygen.
- Aeration: No
- No. of organisms per vessel: 1/replicate/test concentration
- No. of vessels per concentration: 10
- No. of vessels per control: 10
- Biomass loading rate: 1 daphnid per 130 mL of dilution water

TEST MEDIUM / WATER PARAMETERS
- Source: Elendt M4, according to Elendt (1990) was used as a culture medium
- Culture medium different from test medium: No
- Intervals of water quality measurement: The water quality parameters (i.e. pH-values, dissolved oxygen concentration, total hardness and water temperature) were measured once every 7 days, in the fresh (0 hours) and the old media (24 hours), in one replicate of the control and at the highest concentration of the test item. The temperature in the incubator was recorded with a thermohygrograph during the test.

OTHER TEST CONDITIONS
- Adjustment of pH: Not reported
- Photoperiod: 16/8 h light/dark cycle
- Light intensity: Max. 20 µEm(-2)s(-1)

EFFECT PARAMETERS MEASURED: Mortality of parental daphnids and abnormalities (e.g. swimming behaviour, number of male and winter eggs) were observed and recorded at each day of observation. First appearance of juveniles was checked daily. The number of neonates (alive and dead progeny) was counted and dates of release were recorded once a day from the first day of production of neonates until the end of the test. At the end of the test, the total length of each daphnid and the mean dry body weight of the surviving daphnids at each concentration level and control were determined.

RANGE-FINDING STUDY: Yes, a range finding test of 21 days duration was carried out
- Test concentrations: 0, 0.16, 0.4 , 1, 2.5 mg/L (nominal)
- Test design: Four replicates with one daphnid per replicate were tested. The test concentrations were analytical verified two times per week in fresh (0 h) and old media (8 and 24 h)
- Results used to determine the conditions for the definitive study: 100% mortality was observed at 1, 2.5 mg/L while there was no mortality in rest of the test groups. Details are provided in the study report.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.63 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mortality
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.276 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
act. ingr.
Basis for effect:
mortality
Details on results:
- Mortality of parent animals: The test material induced biologically significant adult mortality of 100% in the nominal concentration level of 1.0 mg/L after 21 days. In the concentration levels 0.16 mg/L and 0.63 mg/L, only one of ten parental daphnids did not survive until the end of the test after 21 days, which is not biologically significant. In the control and the concentration levels of 0.25 and 0.40 mg/L all daphnids survived until the end of the test period of 21 days.

- Body length of parent animals: The mean values for body length of the surviving parental daphnids respective to tested nominal concentration levels of 0.16 to 0.63 mg/L were determined to be in the range of 4.98 to 5.23 mm per daphnid and 5.03 mm per daphnid in the control group.

- Body weight of parent animals: The mean dry body weight was determined to be in the range of 0.922 to 1.08 mg per daphnid respective to nominal concentration levels of 0.16 to 0.63 mg/L and 1.12 mg per daphnid in the control

- Time to first brood release: The first brood was released on Days 8 or 9 by all parental daphnids of the control and at the nominal concentration levels of 0.16 and 0.40 mg/L. One of ten parental daphnids at the nominal concentration level of 0.25 mg/L released the first brood on Day 11 and one of nine surviving daphnids released the first brood on Day 13 in the 0.63 mg/L exposure.
In the control and the test levels of 0.16 to 0.63 mg/L 4 broods per surviving parental daphnid were typically observed. One of ten parental daphnids at the concentration level of 0.40 mg/L produced 5 broods. One of nine surviving daphnids at the concentration level of 0.63 mg/L produced 3 broods during the test period

- Reproductive output: The average number of living juveniles per surviving parental daphnid at the end of the test after 21 days was 80 in the control group. The reproductive output in the nominal concentration levels of 0.16 to 0.63 mg/L was comparable to the reproductive output in the control. In the nominal concentration level of 1.0 mg/L, no parental daphnids survived until the end of the study.
The coefficient of variation of the number of living offspring produced per parent was 8 % in the control. In the nominal test concentrations of 0.16 to 0.63 mg/L the coefficient of variation was in the range of 8 to 13 %

- Other biological observations: No males or ephippia (winter eggs) were observed in the control or in the test groups.

Results with reference substance (positive control):
- Results with reference substance valid: Yes
- Relevant effect levels: EC50: 1.73 mg/L (CL: 1.67 - 1.79 mg/L)
The EC50-value of the reference item potassium dichromate after 24 h is within the prescribed concentration range of 1.0 - 2.5 mg/L of quality criteria according to AQS P 9/2 (05/1996) for daphnids clone 5 cultured in Elendt M4 medium. The EC50-value of the reference material is also within the recommended range of 0.6 - 2.1 mg/L according to OECD-Guideline 202.
Reported statistics and error estimates:
- The NOEC and the LOEC for the adult mortality as the most sensitive effect in this study was determined directly from the results based on both nominal and time weighted mean measured concentrations of the test material.
- Significant deviations were determined in comparison to the control using statistical standard procedures as normality test, equal variance test and analysis of variance (parametric Anova, Dunnett’s method).
- Prior to running the parametric ANOVA, a normality test and an equal variance test were performed. P-values of the normality and equal variance tests were 0.05. The alpha-value 0.05.
- For the determination of significant deviations of the reproduction rates and the number of dead juveniles and aborted eggs, One Way Analysis of Variance, DUNNETT’S method (p = 0.05) was used. The coefficients of variation around the mean number of living offspring produced per parent in the control and the test groups were evaluated.

Any other information on results incl. tables

Table 2: Chronic Toxicity Study ofToluene-2,5-Diamine Sulfateto daphnia magna (study # 66355)

Effects

 

Time Weighted Mean Measured Concentrations

(Nominal Concentrations)

[mg/L])

 

Control

0.0620

(0.160)

0.107

(0.250)

0.175

(0.400)

0.276

(0.630)

0.364

(1.00)

Mean Number of Living Juveniles per

Producing Parent (Reproduction Rate ± SD)

80±6

81±8

83±6

89±9

79±10

---

Coefficient of Variation of the Mean Number of Juveniles per Producing Parent [%]

 8

10

 8

10

13

---

Presence of Dead Juveniles

 Present

Present

Present

Present

Present

---

Mean Intrinsic Rates of Natural Increase

 0.43

 0.43

 0.44

 0.43

 0.42

---

Appearance of First Brood [Mean Day]

 8.20

 8.56

 8.70

 8.70

 9.11

---

Mean Number of Broods

 4.00

 4.00

 4.00

 4.10

 3.89

---

Parental Daphnids: Mean Dry Weight [mg]

 1.12

 1.08

 1.08

 1.02

 0.922

---

Parental Daphnids: Mean Body Length [mm]

 5.03

 5.06

 4.98

 5.23

 5.03

---

Adult Mortality after 21 Days [%]

 0

10

 0

 0

10

100

Endpoint

Measured mg/L

Nominal mg/L

NOEC

0.276 mg/L

0.630

LOEC

0.364 mg/L

1.00

---     = Not applicable, due to 100 % adult mortality

SD   = Standard deviation

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The 21 d NOEC of Toluene-2,5-Diamine Sulfate to Daphnia magna was 0.63 mg/L based on mortality. The result is based on nominal concentration.
Executive summary:

The 21 d chronic toxicity of Toluene-2,5-DiamineSulfate dispersion to Daphnia magna was determined under semi-static conditions, following the OECD Guideline 211 (Daphnia magna Reproduction Test) guideline. 

Ten daphnids, individually held, were used per concentration level, and control. Nominal test concentrations were 0,0.160, 0.250, 0.400, 0.630, 1.00 mg/L and the time weighed mean measured test concentrations were 0.0620, 0.107, 0.175, 0.276, 0.364 mg/L. The measured concentrations of the test item were in the range of 94 to 110 % of the nominal values in the fresh media (0 hours) and < LOQM to 41 % in the old media (24 hours).

The test material induced biologically significant adult mortalityof 100% in the nominal concentration level of 1.0 mg/L after 21 days.In all other test concentrations, all daphnids survived or only one of the ten parental daphnids did not survive until the end of the test after 21 days, which is not biologically significant.The average number of living juveniles per surviving parental daphnid at the end of the test was 80 in the control group. The reproductive output in the nominal concentration levels of 0.16 to 0.63 mg/L was comparable to the reproductive output in the control.

No males or ephippia (winter eggs) were observed in the control or in the test groups.

The EC50 of the reference material, potassium dichromate, was1.73 mg/L (95% confidence limits: 1.67 - 1.79 mg/L) after 24 hours and therefore within the prescribed concentration range of 1.0 - 2.5 mg/L of quality criteria according to AQS P 9/2 (05/1996) for daphnids clone 5 cultured in Elendt M4 medium.

The 21 d NOEC of Toluene-2,5-Diamine Sulfate to Daphnia magna was 0.63 mg/L based on mortality. The result is based on nominal concentration.

This chronic toxicity test is classified as acceptable, and satisfies the guideline requirements for the OECD Guideline 211.