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Short-term toxicity to fish

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Reference
Endpoint:
short-term toxicity to fish
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 203 (Fish, Acute Toxicity Test)
Version / remarks:
1992
Deviations:
yes
Remarks:
pH varied during test
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 right before renewal of the test medium (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):
Oryzias latipes
Details on test organisms:
TEST ORGANISM
- Common name: Japanese rice fish or Medaka
- Source: Test organisms were obtained from Takizawa fish farm (Japan), cultured at the Japan Food Research Laboratory
- Age at study initiation: not reported
- Length at study initiation: 2.2 cm (2.0-2.4 cm)
- Weight at study initiation: 0.14 g (0.11-0.20 g)

ACCLIMATION
- Acclimation period: at least 7 days
- Acclimation conditions: Same as test conditions (dechlorinated tap water; T = 24 ± 1 °C; 16:8 h light/dark)
- Type and amount of food during acclimation: Commercially available formulated feed (TetraMin)
- Feeding frequency during acclimation: Not specified, test organisms were not fed 24 h prior to testing
- Health during acclimation (any mortality observed): < 5 % mortality; only healthy and macroscopically normal fish were used for testing


FEEDING DURING TEST
- none
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
44.0 mg/L as CaCO3
Test temperature:
23.5 - 24.2 °C
pH:
4.7 - 7.6
Dissolved oxygen:
6.2 - 9.4 mg/L
Nominal and measured concentrations:
Nominal: 0 (control), 22, 32, 46, 68, 100 mg/L
Measured (at 0 h): < 0.05 (control), 22.6, 32.7, 45.6, 71.4, 105 mg/L
Measured (at 48 h, before water change): < 0.05 (control), 22.6, 32.0, 46.3, 70.7, 104 mg/L. The highest test concentration (nominal = 100 mg/L) was measured at 3 h, as all test organism were dead at this point.
Details on test conditions:
TEST SYSTEM
- Test vessel: 5 L glass tank
- Fill volume: 3 L test solution
- Aeration: None
- Renewal rate of test solution: Every 48 h
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: < 1.0 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tokyo 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:
- mortality (unable to react at stimulus), every 24 h
- abnormal respiration, every 24 h
- abnormal swimming, every 24 h
- ability to swim, every 24 h

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.4-1.5
- Range finding study: yes
- Test concentrations: 100 mg/L, geometric progression with a factor of 10
Reference substance (positive control):
yes
Remarks:
Copper sulfate pentahydrate
Duration:
96 h
Dose descriptor:
LC0
Effect conc.:
68 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
82 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: Effects on survival at 100 mg/L are considered to be associated with an acidification of the test medium.
Details on results:
No visible abnormalities (abnormal respiration, abnormal swimming) were observed for test concentrations up to 68 mg/L during 96 h of exposure. No observations were made at 100 mg/L as all test organisms were dead at the point of observation. Measured concentrations were at 99-105 % of the nominal values at 0 h and at 48 h (prior to renewal of test medium).

During the testing period the test medium pH ranged from 6.3-7.6 for the controls and the treatment groups with m-toluic acid concentrations of ≤ 46 mg/L. At 68 mg/L the pH of the test medium was, except for two time points (5.6-5.8), around 6.1-6.9. However, at 100 mg/L the pH was 4.7 and dropped below the recommended pH range of 6.0-8.5 (OECD 203, 1992).

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 203 (1992), if the pH does not remain in the range 6.0-8.5, 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 100 mg/L m-toluic acid dropped below the recommended pH range of 6.0-8.5 (OECD 203, 1992). Thus, effects on survival of Oryzia latipes observed at 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 96 h LC50 of 82 mg/L is derived from this test.
Results with reference substance (positive control):
96 h LC50: 0.69 mg/L
Reported statistics and error estimates:
The statistical method for calculation of LC50 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 fresh

48 h old

mean

0 h fresh

48 h old

Control

< 0.05

< 0.05

-

-

-

22

22.6

22.6

22.6a

103

103

32

32.7

32.0

32.3t

102

100

46

45.6

46.3

46.0a

99

101

68

71.4

70.7

71.0t

105

104

100

105

104*

104t

105

104*

a: arithmetic mean

t: time-weighted mean

* measurements after 3 h, as all test organisms were dead at 48 h

Table 2: Mortality of Oryzias latipes

Nominal conc. (mg/L)

Cumulative number of dead (Percent mortality)

24 h

48 h

72 h

96 h

Control

0 (0)

0 (0)

0 (0)

0 (0)

22

0 (0)

0 (0)

0 (0)

0 (0)

32

0 (0)

0 (0)

0 (0)

0 (0)

46

0 (0)

0 (0)

0 (0)

0 (0)

68

0 (0)

0 (0)

0 (0)

0 (0)

100

10 (100)

10 (100)

10 (100)

10 (100)

Table 3: Visible abnormalities during the exposure period

Nominal conc. (mg/L)

Symptoms

24 h

48 h

72 h

96 h

Control

n.a.d.

n.a.d.

n.a.d.

n.a.d.

22

n.a.d.

n.a.d.

n.a.d.

n.a.d.

32

n.a.d.

n.a.d.

n.a.d.

n.a.d.

46

n.a.d.

n.a.d.

n.a.d.

n.a.d.

68

n.a.d.

n.a.d.

n.a.d.

n.a.d.

100

-*

-*

-*

-*

n.a.d.: no abnormalities detected

* No observations could be made as all test organisms were dead at this point

Table 4: pH values of test solutions over the 96 h test period

 

pH

Hours \ Nominal conc. (mg/L)

Control

22

32

46

68

100

0

7.6

7.0

6.8

6.5

5.8

4.7

24

7.3

7.0

6.9

6.7

6.1

4.7*

48 old

7.5

7.2

7.1

7.0

6.7

-

48 fresh

7.6

6.9

6.7

6.3

5.6

-

72

7.4

7.2

7.1

7.0

6.5

-

96

7.4

7.5

7.4

7.4

6.9

-

* Measurement at 3 h, as all test organisms were dead at this point

Table 5: Dissolved oxygen (DO) concentrations over the 96 h test period

 

DO (mg/L)

Hours \ Nominal conc. (mg/L)

Control

22

32

46

68

100

0

9.4

9.0

9.0

9.0

9.0

8.9

24

7.4

7.5

7.7

7.5

7.6

8.4*

48 old

6.9

6.7

6.8

7.0

7.1

-

48 fresh

8.9

8.9

8.9

8.9

8.8

-

72

7.6

7.2

7.5

7.2

7.3

-

96

6.9

6.7

6.6

6.4

6.2

-

* Measurement at 3 h, as all test organisms were dead at this point

Table 6: Temperature values over the 96 h test period

 

Temperature (°C)

Hours \ Nominal conc. (mg/L)

Control

22

32

46

68

100

0

23.8

23.6

23.6

23.5

23.5

23.5

24

24.2

23.9

24.0

24.0

24.0

23.5*

48 old

23.5

23.6

23.6

23.6

23.5

-

48 fresh

24.2

24.1

24.1

24.0

24.0

-

72

24.2

23.9

23.9

23.9

23.9

-

96

23.8

24.0

24.1

24.1

24.1

-

* Measurement at 3 h, as all test organisms were dead at this point

Validity criteria fulfilled:
yes
Conclusions:
Oryzias latipes (length: 2.0-2.4 cm) were exposed to m-toluic acid in dechlorinated tap water in an acute toxicity test according to OECD TG 203. The mortality of Japanese rice fish was determined after 24, 48, 72 and 96 h and an effect of exposure to m-toluic acid was observed after 96 h; i.e. all fish were dead at 100 mg/L whereas mortality was not observed at 68 mg/L nominal)). At 100 mg m-toluic acid/L, the solution pH decreased to a pH of 4.7. 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 203 (1992), if the pH does not remain in the range 6.0-8.5, 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 the solution pH was not adjusted, the pH at 100 mg/L m-toluic acid dropped below the recommended pH range of 6.0-8.5 (OECD TG 203). Thus, effects on survival of Oryzia latipes 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 96-h LC50 of 82 mg/L is derived conservatively for m-toluic acid.

Description of key information

In a GLP-conform OECD 203 acute toxicity test on Oryzias latipes (Japanese rice fish or Medaka), effects on survival were observed at 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, a 96-h LC50 of 82 mg/L is derived conservatively for m-toluic acid.

Key value for chemical safety assessment

LC50 for freshwater fish:
82 mg/L

Additional information