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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
1984
Deviations:
yes
Remarks:
in some treatments pH was below 6-9 and pH in stock solution was not adjusted to that of the test medium prior to testing
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
50 ml water samples for concentration measurement of m-toluic acid were taken at test initiation (0 h) and at the end of the test (48 h).
Vehicle:
no
Details on test solutions:
A stock solution was prepared by dissolving m-toluic acid in dechlorinated tap water. This stock solution was diluted to prepare appropriate test solutions.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Source: Test organisms were obtained from the National Institute for Environmental Studies (Japan)
- Age of parental stock: 2-4 weeks
- Age at test initiation: < 24 h
- Feeding during test: None

ACCLIMATION
- Acclimation period: 2-4 weeks
- Acclimation conditions: same as test (dechlorinated tap water; T: 20 ± 1 °C; 16:8 h light/dark)
- Type and amount of food: Chlorella vulgaris; 0.15 mg organic carbon per daphnid per day
- Health during acclimation (any mortality observed): Only containers in which no abnormalities and mortality were observed, were used for testing
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
72 mg/L as CaCO3
Test temperature:
19.6-20.9 °C
pH:
3.9-7.8 (see additional results)
Dissolved oxygen:
8.6-9.5 mg/L
Nominal and measured concentrations:
Nominal: 0 (control), 10, 18, 32, 56, 100, 180, 320 mg/L
Measured (at 0 h): < 0.05 (control), 11.9, 21, 26.5, 58.6, 107, 188, 318 mg/L
Measured (at 48 h): < 0.05 (control), 11.6, 20.7, 26.5, 59.6, 106, 178, 328 mg/L. At nominal concentrations of 180 and 320 mg/L, measurements were conducted at 24 h instead of 48 h, since all daphnids were dead at 48 h.
Details on test conditions:
TEST SYSTEM
- Test vessel: 270 ml glass beaker
- Type: closed
- Fill volume: 100 ml test solution

- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tsukuba City tap water was treated with activated charcoal, residual chlorine was removed and it was fully ventilated

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16:8 h light:dark
- Light intensity: not reported

EFFECT PARAMETERS MEASURED:
- immobilization, every 24 h

RANGE-FINDING STUDY
- Performed prior to definitive test, concentrations not specified
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
75 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: Effects on mobility of D. magna observed at or above 100 mg/L are considered to be associated with an acidification of the test medium.
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
75 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: Effects on mobility of D. magna observed at or above 100 mg/L are considered to be associated with an acidification of the test medium
Details on results:
Measured concentrations were at 83-119 % of the nominal values at 0 h and at 48 h. pH values decreased significantly with increasing concentrations of the test substance m-toluic acid. At test concentrations of ≥ 100 mg m-toluic acid/L the test medium pH dropped below the pH range of 6-9 specified in OECD test guideline 202.

According to the ECHA guidance on IR & CSA Chapter R.7b: Endpoint specific guidance (Version 4.0, June 2017), where a substance causes a change in pH of the test medium (e.g. strong acids and bases), the pH should be adjusted to lie within the specified range for the test using a suitable technique. Furthermore, according to OECD 202 (2004), if the pH does not remain in the range 6-9, then a second test could be carried out, adjusting the pH of the stock solution to that of the dilution water before addition of the test substance. Since no precautions were taken prior to testing to adjust the pH of the test substance e.g. in the stock suspension, the pH of the test medium in treatment groups with m-toluic acid concentration equal to or above 100 mg/L dropped below the recommended pH range of 6-9 (OECD 202, 2004). Thus, effects on mobility of D. magna observed at or above 100 mg/L are considered to be associated with an acidification of the test medium and are not considered to be reliable. However, conservatively for toluic acid an 48 h EC50 of 75 mg/L is derived from this test.
Results with reference substance (positive control):
48 h EC50: 0.42 mg/L (Test conducted in October 1998)
48 h EC50: 0.44 mg/L (Test conducted in June 1999)
EC50 values obtained for the reference substance potassium dichromate are slightly lower than the values specified in the OECD guideline 202 (EC50: 0.6-2.1 mg/L), which might indicate a slightly increased sensitivity of test organisms in the present study.
Reported statistics and error estimates:
The statistical method for calculation of EC50 values was binomial.

Table 1: Nominal and measured concentrations at 0 and 48 h

Nominal conc. (mg/L)

Measured conc. (mg/L)

Percent of nominal

0 h

48 h

mean

0 h

48 h

Control

< 0.05

< 0.05

-

-

-

10

11.9

11.6

11.7t

119

116

18

21.0

20.7

20.8t

117

115

32

26.5

26.5

26.5a

83

83

56

58.6

59.6

59.1a

105

106

100

107

106

106t

107

106

180

188

178*

183t

104

99*

320

318

328*

323a

99

103*

a: arithmetic mean

t: time-weighted mean

* measurements after 24 h, as all test organisms were dead at 48 h

Table 2: Immobility of Daphnia magna

Nominal conc. (mg/L)

Cumulative number of immobile or dead organisms (Percent immobility)

24 h

48 h

Control

0 (0)

0 (0)

10

0 (0)

0 (0)

18

0 (0)

0 (0)

32

0 (0)

0 (0)

56

0 (0)

0 (0)

100

20 (100)

20 (100)

180

20 (100)

20 (100)

320

20 (100)

20 (100)

Table 3: pH values of test solutions at 0 h and 48 h

Nominal conc. (mg/L)

pH

0 h

48 h

Control

7.8

7.7

10

7.3

7.7

18

7.1

7.7

32

7.0

7.6

56

6.7

7.5

100

5.4

6.3

180

4.4

4.3*

320

4.0

3.9*

* Measurement at 24 h, as all test organisms were dead at this point

Table 4: Dissolved oxygen (DO) concentrations at 0 h and 48 h

Nominal conc. (mg/L)

DO (mg/L)

0 h

48 h

Control

9.5

8.8

10

9.4

8.6

18

9.5

8.6

32

9.4

8.6

56

8.9

8.6

100

8.9

8.7

180

9.1

8.9*

320

8.9

8.9*

* Measurement at 24 h, as all test organisms were dead at this point

Table 5: Temperature values at 0 h and 48 h

Nominal conc. (mg/L)

Temperature (°C)

0 h

48 h

Control

20.1

19.7

10

20.3

20.7

18

20.2

20.6

32

20.2

20.8

56

20.3

20.9

100

20.2

20.8

180

20.3

19.6*

320

20.3

19.7*

* Measurement at 24 h, as all test organisms were dead at this point

Validity criteria fulfilled:
yes
Conclusions:
Daphnia magna neonates (< 24 h old) were exposed to m-toluic acid in dechlorinated tap water in an acute immobilisation test according to OECD TG 202. The mobility of Daphnia magna was determined after 24 and 48 h and toxicity was not observed (24-h EC50: > 56.0 mg/L (nominal), 48-h EC50: > 56.0 mg/L (nominal)). A significant decrease of pH with increasing test concentrations led to the pH being below the recommended range of 6-9 (OECD TG 202) at test concentrations ≥ 100 mg/L. Immobilizing effects were also observed at concentrations ≥ 100 mg/L, with an increase from 0 % immobilization at 56 mg/L to 100 % immobilization at 100 mg/L. According to the ECHA guidance on IR & CSA Chapter R.7b: Endpoint specific guidance (Version 4.0, June 2017), if a substance causes a change in pH of the test medium (e.g. strong acids and bases), the pH should be adjusted to remain within the specified range for the test using a suitable technique. Furthermore, according to OECD TG 202, if the pH does not remain in the range 6-9, then a second test could be carried out, adjusting the pH of the stock solution to that of the dilution water before addition of the test substance.The pH of solutions containing the test substance was, however, not adjusted. Thus, effects on mobility of D. magna observed at ≥ 100 mg/L are considered to be associated with an acidification of the test medium and are not considered to be related to the intrinsic toxicity of m-toluic acid. However, a 48-h EC50 of 75 mg/L is conservatively derived for m-toluic acid.

Description of key information

In a GLP-conform OECD 202 acute immobilisation test on Daphnia magna, effects on mobility of D. magna were observed at or above 100 mg/L. These effects are associated with an acidification of the test medium and are not considered to be related to the intrinsic toxicity of m-toluic acid. However, conservatively a 48-h EC50 of 75 mg/L m-toluic acid is derived.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
75 mg/L

Additional information