2-nitrotoluene

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

Basic data given. Restrictions of the method are: - Test period only 4 instead of 48h (represents only 8% of the in the OECD and other current guidelines suggested test period)-Temperature during the test was higher (25 °C) than the one suggested by OECD and other current guidelines (22 °C) - Yeast is not a standard food for Daphnia

Justification for type of information:

Due to methodological deficiencies, this study was rated with a Klimisch score of 3 (‘not reliable’). As none of the available studies can serve as a key study with a sufficiently detailed reporting justifying a Klimisch score of 1 or 2 for the given endpoint, several independent sources were used in a weight of evidence approach. ‘Using weight of evidence implies that no single study of sufficient quality and reliability exists, and that information from several independent sources is required to be able to reach a conclusion on a particular property of the substance.’ (guidance document How to prepare registration and PPORD dossiers, p. 87). Despite being considered not  reliable (Klimisch 3), the experimental value obtained in this study still supports the WoE conclusion confirming that the EC50 is in the order of magnitude of the concluded key value.

Principles of method if other than guideline:

not specified

GLP compliance:

not specified

Specific details on test material used for the study:

purity more than 98%, purchased from E. Merck, Darmstadt, Germany.

Test organisms (species):

Daphnia pulex

Details on test organisms:

Daphnia (Daphnia pulex) was obtained from Plant Toxicology Department, Taiwan Agricultural Chemicals and Toxic Substances Research Institute, Taichung, Taiwan.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

4 h

Test temperature:

25 ± 1°C

pH:

6.6

Dissolved oxygen:

4.9 mgL/L

Salinity:

hardness 215 mg/L as CaCO3

Details on test conditions:

Daphnia were fed with yeast in a 224 (diameter) x 46 (height)-cm circular plastic pool. Before the experiment, the test organisms were acclimatized in aquaria for 2 weeks under conditions similar to those under which the tests were performed.

Twenty daphnia, within 24 hr after hatching, were introduced in each 250-mL beaker containing 100 mL of test chemical solution with different concentrations. Adequate
concentrations of 2-nitrotoluene were prepared individually in duplicate for each test chemical. Mortality of daphnia was observed after an incubation period of 4 h. If the appropriate concentration for mortality was beyond the test range, the experiment was carried out once more with a more appropriate concentration.

Duration:

4 h

Dose descriptor:

LC50

Effect conc.:

101.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Validity criteria fulfilled:

not specified

Remarks:

Restrictions of the method: - Test period only 4 instead of 48h -Temperature during the test was higher (25 °C) than 22 °C - Yeast is not a standard food for Daphnia

Executive summary:

Yen, 2002: In a short-term toxicity test on Daphnia pulex an LC50 value of 1.012 mg/l after a 48-h exposure period was obtained for 2-nitrotoluene. After request, one of the authors (Wang 2003) communicated that the reported values are below the observed values by a factor of 100. Thus, the correct LC50 is 101.2 mg/l. Despite being considered not  reliable (Klimisch 3, Restrictions of the method: - Test period only 4 instead of 48h -Temperature during the test was higher (25 °C) than 22 °C - Yeast is not a standard food for Daphnia), the experimental value obtained in this study still supports the WoE conclusion confirming that the EC50 is in the order of magnitude of the concluded key value. 

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Remarks:

National Guideline study with acceptable restrictions

Justification for type of information:

Due to insufficient reporting of the experimental details, this study was rated with a Klimisch score of 4 (‘not assignable’). As none of the available studies can serve as a key study with a sufficiently detailed reporting justifying a Klimisch score of 1 or 2 for the given endpoint, several independent sources were used in a weight of evidence approach. ‘Using weight of evidence implies that no single study of sufficient quality and reliability exists, and that information from several independent sources is required to be able to reach a conclusion on a particular property of the substance.’ (guidance document How to prepare registration and PPORD dossiers, p. 87)

Qualifier:

according to guideline

Guideline:

other: protocol of the Dutch Standards Organisation, NEN 6501 (1980)

GLP compliance:

not specified

Specific details on test material used for the study:

- Name of test material (as cited in study report): 2-nitrotoluene
- Analytical purity: > 99%

Test organisms (species):

Daphnia magna

Details on test organisms:

- daphnids were fed on 1x10E8 cells/l of Chlorella pyrenoidosa

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

11 mg/L

Remarks on result:

other: 95% CL: 5.6-18

Details on results:

CL=confidence limits

Validity criteria fulfilled:

not specified

Executive summary:

Maas-Diepeveen, 1986: In a short-term toxicity tests on crustaceans (D. magna) in analogy with the Dutch national standard method NEN6501 an LC50 value of 11 mg/l after a 2 days exposure period was obtained.

Reference 3

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

other information

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

The exposure time was too short (24 h instead of the required 48h)

Justification for type of information:

Due to methodological deficiencies, this study was rated with a Klimisch score of 3 (‘not reliable’). As none of the available studies can serve as a key study with a sufficiently detailed reporting justifying a Klimisch score of 1 or 2 for the given endpoint, several independent sources were used in a weight of evidence approach. ‘Using weight of evidence implies that no single study of sufficient quality and reliability exists, and that information from several independent sources is required to be able to reach a conclusion on a particular property of the substance.’ (guidance document How to prepare registration and PPORD dossiers, p. 87). Despite being considered not  reliable (Klimisch 3), the experimental value obtained in this study still supports the WoE conclusion confirming that the EC50 is in the order of magnitude of the concluded key value.

Principles of method if other than guideline:

- Immobilization test according to Bringmann G, Kuehn R (1982)

GLP compliance:

not specified

Specific details on test material used for the study:

- Name of test material (as cited in study report): 2-Nitrotoluene
- Analytical purity: not specified

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

- stock solution: test substance was dissolved by stirring in test medium
- pH did not change
- duplicate dilution series were prepared in test medium

Test organisms (species):

Daphnia magna

Details on test organisms:

- 10 daphnia (strain: IRCHA; = 24 h old) per concentration

Test type:

static

Total exposure duration:

24 h

Test temperature:

20°C

pH:

initial pH 8.0 +/- 0.2 (not adjusted during exposure)

Dissolved oxygen:

prior to testing test medium was aerated until oxygen saturation

Duration:

24 h

Dose descriptor:

EC0

Effect conc.:

6.8 mg/L

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

16 mg/L

Remarks on result:

other: P 95%. 14-18

Duration:

24 h

Dose descriptor:

EC100

Effect conc.:

26 mg/L

Validity criteria fulfilled:

not specified

Executive summary:

Bringmann and Kühn (1982) carried out an acute short test on Daphnia magna. The effects of 2-nitrotoluene were measured on 24-h individuals after 24-h exposure period. Following results are provided: an EC0 of 6.8 mg/l, an EC50 of 16 mg/l (14-18 mg/l) and an EC100 of 26 mg/l. This result has been included as additional information. Despite being considered not  reliable (Klimisch 3, the exposure time was too short: 24 h instead of the required 48h), the experimental value obtained in this study still supports the WoE conclusion confirming that the EC50 is in the order of magnitude of the concluded key value.

Reference 4

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

The exposure time was too short (24 h instead of the required 48h)

Justification for type of information:

Due to methodological deficiencies, this study was rated with a Klimisch score of 3 (‘not reliable’). As none of the available studies can serve as a key study with a sufficiently detailed reporting justifying a Klimisch score of 1 or 2 for the given endpoint, several independent sources were used in a weight of evidence approach. ‘Using weight of evidence implies that no single study of sufficient quality and reliability exists, and that information from several independent sources is required to be able to reach a conclusion on a particular property of the substance.’ (guidance document How to prepare registration and PPORD dossiers, p. 87). Despite being considered not  reliable (Klimisch 3), the experimental value obtained in this study still supports the WoE conclusion confirming that the EC50 is in the order of magnitude of the concluded key value.

Principles of method if other than guideline:

- Immobilization test according to Bringmann G, Kuehn R (1977)

GLP compliance:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): 2-Nitrotoluene
- Analytical purity: not specified

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

- stock solution: test substance was dissolved by stirring in tap water
- pH did not change
- triple dilution series were prepared in tap water

Test organisms (species):

Daphnia magna

Details on test organisms:

- 10 daphnia (= 24 h old) per concentration

Test type:

static

Total exposure duration:

24 h

Test temperature:

20-22°C

Duration:

24 h

Dose descriptor:

LC0

Effect conc.:

13 mg/L

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

42 mg/L

Duration:

24 h

Dose descriptor:

LC100

Effect conc.:

100 mg/L

Validity criteria fulfilled:

not specified

Executive summary:

Bringmann and Kühn (1977) carried out an acute short test on Daphnia magna. The effects of 2-nitrotoluene were measured on 24-h individuals after 24-h exposure period. Following results are provided: an LC0 of 13 mg/l, an LC50 of 42 mg/l and an LC100 of 100 mg/l. This result has been included as additional information. Despite being considered not  reliable (Klimisch 3, the exposure time was too short: 24 h instead of the required 48h), the experimental value obtained in this study still supports the WoE conclusion confirming that the EC50 is in the order of magnitude of the concluded key value.

Reference 5

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Remarks:

Guideline study with acceptable restrictions

Justification for type of information:

Due to insufficient reporting of the experimental details, this study was rated with a Klimisch score of 4 (‘not assignable’). As none of the available studies can serve as a key study with a sufficiently detailed reporting justifying a Klimisch score of 1 or 2 for the given endpoint, several independent sources were used in a weight of evidence approach. ‘Using weight of evidence implies that no single study of sufficient quality and reliability exists, and that information from several independent sources is required to be able to reach a conclusion on a particular property of the substance.’ (guidance document How to prepare registration and PPORD dossiers, p. 87)

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

GLP compliance:

not specified

Specific details on test material used for the study:

- Name of test material (as cited in study report): 2-nitrotoluene
- Analytical purity: >99%
- Supplier: Fluka AG (Buchs, Switzerland)

Analytical monitoring:

no

Details on test solutions:

- Stock solutions were made, 24 h before the experiments, directly in DSW (dutch standard water) and stirred until use.
- Test solutions were freshly prepared, by dilution of the stock solution in DSW, immediately before each test.
- 5 concentration levels (and one control) were tested with a dilution factor of 2.
- Test concentration ranges were attempted to be from 4xEC50 to EC50/4.
- The highest test concentrations were kept below solubility.

Test organisms (species):

Daphnia magna

Details on test organisms:

- Age at study initiation (mean and range, SD): 24 h

breeding:
- temperature: 20 ± 1 °C
- 12 h photoperiod
- dutch standard water (DSW)
- three times per week 2/3 of the water was renewed
- animals were fed daily with 6 ml of a Chlorella vulgaris solution
- young and dead animals were removed daily

Test type:

static

Water media type:

freshwater

Total exposure duration:

48 h

Test temperature:

18-20 °C (± 1 °C)

pH:

8.0-8.3

Dissolved oxygen:

8.1-9.7 mg/L

Details on test conditions:

- culture water (DSW) was used as test water
- test vessels: 50 ml beakers
- Photoperiod: 12 h
- animals were starved from 24 h before the tests until the end
- 10 daphnids (24 h-old) were exposed, in duplicate, to 40 ml test solution
- temperature, pH and oxygen levels were measured before and after each test solution renewal

Reference substance (positive control):

not specified

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

13.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL: 12.0-14.5

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

12.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL: 10.9-13.9

Details on results:

CL=confidence limits

Validity criteria fulfilled:

not specified

Executive summary:

Ramos et al. (1998) performed a toxicity tests according to the OECD guidelines for testing chemicals. The authors provided a 24h-EC50 of 13.2 mg/l (12.0 -14.5 mg/l) and a 48h-EC50 of 12.3 mg/l (10.9 -13.9 mg/l) on Daphnia magna.

Reference 6

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Remarks:

National Guideline study with acceptable restrictions

Justification for type of information:

Due to insufficient reporting of the experimental details, this study was rated with a Klimisch score of 4 (‘not assignable’). As none of the available studies can serve as a key study with a sufficiently detailed reporting justifying a Klimisch score of 1 or 2 for the given endpoint, several independent sources were used in a weight of evidence approach. ‘Using weight of evidence implies that no single study of sufficient quality and reliability exists, and that information from several independent sources is required to be able to reach a conclusion on a particular property of the substance.’ (guidance document How to prepare registration and PPORD dossiers, p. 87)

Qualifier:

according to guideline

Guideline:

other: protocol of the Dutch Standards Organisation, NEN 6501 (1980)

GLP compliance:

not specified

Specific details on test material used for the study:

- Name of test material (as cited in study report): 2-Nitrotoluene
- Analytical purity: not specified

Analytical monitoring:

not specified

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- species: Daphnia magna
- Age at study initiation (mean and range, SD): < 24 hours
- Feeding during test
- Food type: Chlorella pyrenoidosa

Test type:

static

Water media type:

freshwater

Total exposure duration:

48 h

Hardness:

200 mg/l as CaCO3

Test temperature:

20 +/- 0.5°C

pH:

8.4 +/- 0.1

Dissolved oxygen:

synthetic test medium was saturated with air prior to use

Details on test conditions:

- illumination: 12 h/d

Duration:

48 h

Dose descriptor:

IC50

Effect conc.:

10.9 mg/L

Nominal / measured:

not specified

Conc. based on:

test mat.

Basis for effect:

mobility

Validity criteria fulfilled:

yes

Remarks:

validity criteria were fulfilled during the test: - oxygen content of all solutions did not decrease below 7.9 mg/l (85%) - mortality in the controls never exceeded 10%

Conclusions:

In a short-term toxicity tests on crustaceans (D. magna) in analogy with the Dutch national standard method NEN6501 an IC50 (immobilisation) value of 10.9 mg/l after a 2 days exposure period was obtained.

Executive summary:

Deneer et al., (1989) performed a 48-h immobilisation test for D. magna in static test conditions, according to the protocol of the Dutch Standards Organization, NEN 6501 (1980). During the tests, daphnids were fed with Chlorella pyrenidosa, which at the start of the experiments was present at a concentration of 1·108 cells/l. All daphnids used were <24 hours old at the beginning of the experiments. A 48h-EC50 of 10.9 mg/l was determined. Another value of 21d-IC50 of 7.4 mg/l for Daphnia magna was also established.

Reference 7

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Remarks:

Comparable to guideline study, Basic data given

Justification for type of information:

Due to insufficient reporting of the experimental details, this study was rated with a Klimisch score of 4 (‘not assignable’). As none of the available studies can serve as a key study with a sufficiently detailed reporting justifying a Klimisch score of 1 or 2 for the given endpoint, several independent sources were used in a weight of evidence approach. ‘Using weight of evidence implies that no single study of sufficient quality and reliability exists, and that information from several independent sources is required to be able to reach a conclusion on a particular property of the substance.' (guidance document How to prepare registration and PPORD dossiers, p. 87)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

=> proposal to short-term toxicity tests performed on crustaceans (Daphnia magna) (1979)

GLP compliance:

not specified

Specific details on test material used for the study:

- Name of test material (as cited in study report): 2-Nitrotoluene
- Analytical purity: >99.5
- Supplier: Fluka AG

Analytical monitoring:

yes

Details on sampling:

All test results were corrected for the actual concentration during the test.

Test organisms (species):

Daphnia magna

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

5.4 mg/L

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

8.8 mg/L

Conc. based on:

test mat.

Basis for effect:

mobility

Validity criteria fulfilled:

not specified

Conclusions:

In a short-term toxicity tests on crustaceans (D. magna) in analogy with the OECD 202 proposal (1979) an EC 50 (immobilisation) value of 5.4 mg/l after 48h-exposure period was obtained.

Executive summary:

Canton et al. (1985) carried out short-term toxicity tests on crustaceans (D. magna) in analogy with the OECD 202 proposal (1979). The authors provided a LC50 (immobilisation) and an EC 50 (immobilisation) value of 8.8 mg/l and 5.4 mg/l respectively after a 48h-exposure period.

Description of key information

In order to fulfill the data requirements a weight of evidence (WoE) approach (REACh Regulation (EC) No. 1907/2006, Annex XI Section 1.2) was chosen. This approach is a possible adaptation to the standard information requirements which ‘should be undertaken to maximise the use of existing data and minimise the commissioning of new in vivo testing’ (ECHA Guidance R.7a, 2017, p. 364). The WoE approach serves to reduce or avoid animal testing, which should only be performed as last resort (REACh, article 25). The individual study reports were conducted in accordance with standardized guidelines. The study reports nevertheless show some deficiencies with respect to the comprehensiveness of the presented experimental details. Therefore, a reliability of 1 or 2 and thus adequacy as key study was not assignable for any of the individual studies. Consequently, the studies were assigned a Klimisch score of 4 (according to guidance document R.4, ‘not assignable: studies or data […] which do not give sufficient experimental details […]). Collectively, these experimental studies can be used to conclude on this endpoint and to satisfy the information requirement. The studies by Bringmann et al. were part of the BUA report 1989 and EU risk assessment 2008 which are regarded as peer-reviewed source.

The following studies were chosen for WoE:
Canton et al. (1985) carried out short-term toxicity tests on crustaceans (D. magna) in analogy with the OECD 202 proposal (1979). The authors provided a LC50 (immobilisation) and an EC 50 (immobilisation) value of 8.8 mg/l and 5.4 mg/l respectively after a 48h exposure period. The study was also cited in the BUA report 1989. 

Deneer et al. (1989) performed a 48-h immobilisation test for D. magna in static test conditions, according to the protocol of the Dutch Standards Organization, NEN 6501 (1980). During the tests, daphnids were fed with Chlorella pyrenidosa, which at the start of the experiments was present at a concentration of 1·108 cells/l. All daphnids used were <24 hours old at the beginning of the experiments. A 48h-EC50 of 10.9 mg/l was determined. Another value of 21d-IC50 of 7.4 mg/l for Daphnia magna was also established.

Ramos et al. (1998) performed a toxicity tests according to the OECD guidelines for testing chemicals. The authors provided a 24h-EC50 of 13.2 mg/l (12.0 -14.5 mg/l) and a 48h-EC50 of 12.3 mg/l (10.9 -13.9 mg/l) on Daphnia magna.

Maas-Diepeveen et al. (1986): In a short-term toxicity tests on crustaceans (D. magna) in analogy with the Dutch national standard method NEN6501 an LC50 value of 11 mg/l after a 2 days exposure period was obtained.

Yen, 2002: In a short-term toxicity test on Daphnia pulex an LC50 value of 1.012 mg/l after a 4-h exposure period was obtained for 2-nitrotoluene. After request, one of the authors (Wang 2003) communicated that the reported values are below the observed values by a factor of 100. Thus, the correct 4h-LC50 is 101.2 mg/l. Despite being considered not  reliable (Klimisch 3, Restrictions of the method: - Test period only 4 instead of 48h -Temperature during the test was higher (25 °C) than 22 °C - Yeast is not a standard food for Daphnia), the experimental value obtained in this study still supports the WoE conclusion confirming that the EC50 is in the order of magnitude of the concluded key value. 

The results obtained by Bringmann and Kühn (1977 and 1982) were  included in the WoE approach despite being considered not  reliable (Klimisch 3, the exposure time was too short: 24 h instead of the required 48h) due to methodological deficiencies. The experimental values obtained in these studies still support the WoE conclusion confirming that the EC50 is in the order of magnitude of the concluded key value.

Bringmann and Kühn (1977) carried out an acute short test on Daphnia magna. The effects of 2-nitrotoluene were measured on 24-h individuals after 24-h exposure period. Following results are provided: an LC0 of 13 mg/l, an LC50 of 42 mg/l and an LC100 of 100 mg/l. This result has been included as additional information. 

Bringmann and Kühn (1982) carried out an acute short test on Daphnia magna. The effects of 2-nitrotoluene were measured on 24-h individuals after 24-h exposure period. Following results are provided: an EC0 of 6.8 mg/l, an EC50 of 16 mg/l (14-18 mg/l) and an EC100 of 26 mg/l. 

Conclusion
The EC50 values obtained from these studies are in a similar order of magnitude (48h EC50/LC50 range: 5.4-12.3 mg/L; 24h EC50: 16-42 mg/L). The most conservative 48h-EC50 value (immobilization) of  5.4 mg/L (Canton, 1985) is chosen as key value for assessment.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

5.4 mg/L