4-nitrotoluene

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Basic data given

Principles of method if other than guideline:

other: no guideline followed, see test conditions

GLP compliance:

not specified

Analytical monitoring:

no

Test organisms (species):

Scenedesmus pannonicus

Test type:

static

Water media type:

freshwater

Total exposure duration:

96 h

Test temperature:

23 +/- 2 °C

Details on test conditions:

- ca. 1.5 * 10E+6 organisms (in 3-fold) per group
- 0.15 l Testvolume (per group)
- Lighting (13 W/m2)
- Culturing media (1 L bidest. containing 35 mg/L CaCl2*2H2O, 75 mg/L MgSO4*7H2O, 52 mg/L K2HPO4, 6 mg/l citric acid, 500 mg/L NaNO3, 54 mg/L Na2CO3*10H2O, 6 mg/L Ferricitrate, 330 mg/L NH4NO3, 1 ml (0.0006 g Na2MoO4*2H2O, 2.9 g H3BO3, 0.11 g ZnCl2, 0.08 g CuSO4*5H2O, 0.018 g (NH4)6Mo7O24)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Executive summary:

OECD SIDS (2003):

In a toxicity test with the algae Scenedesmus pannonicus a 96 h-NOEC of 10 mg/l was observed for the endpoint biomass (nominal concentration) (Slooff and Canton, 1983).

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

secondary literature

Remarks:

Although it is stated that the cited result was obtained from Scenedesmus pannonicus, the literature cited contained data only on Scenedesmus quadricauda

Principles of method if other than guideline:

other: Cell multiplication inhibition test according to Bringmann & Kuehn (1977)

GLP compliance:

not specified

Specific details on test material used for the study:

- Name of test material (as cited in Bringmann (1978)): 4-Nitrotoluol
- Analytical purity (as given in Bringmann (1978)): no data

Analytical monitoring:

no

Test organisms (species):

Scenedesmus quadricauda

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

8 d

Test temperature:

27 °C

pH:

7.0

Details on test conditions:

Bringmann & Kuehn (1977):
- 100 ml test solution (neutralized) of certain dilutions of the test substance prepared in replicate
- Test solution contains nutrient medium (all media for test and culture are described in detail in the reference)
- Incubation of 10 ml aliquots in Kapsenberg-culture vials in artifical continuous light
- Cell multiplication measured turbidimetrically at 578 nm with an optical path length of 10 mm

Duration:

8 d

Dose descriptor:

other: TGK

Effect conc.:

15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Executive summary:

The cell growth inhibition test with the unicellular green alga Scenedesmus quadricauda gave the following results (Bringmann and Kuehn, 1977): TTC (= EC3) 15 mg/L after 8 days.

Reference 3

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Documentation is insufficient for assessment. It is unclear whether the algae are within the exponential growth throughout the whole exposure period of 8 days.

Principles of method if other than guideline:

other: cell multiplication inhibition test according to Bringmann (1975)

GLP compliance:

no

Specific details on test material used for the study:

- Name of test material (as cited in publications): 4-Nitrotoluol
- Analytical purity: no data

Analytical monitoring:

no

Test organisms (species):

Microcystis aeruginosa

Details on test organisms:

- Source (laboratory, culture collection): Institute of Plant Physiology, University of Göttingen

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

8 d

Test temperature:

27 °C

pH:

7.0

Details on test conditions:

- relative humidity: 50%
- 100 ml test solution (neutralized) of certain dilutions of the test substance prepared in replicate
- Test solution contains nutrient medium (all media for test and culture are described in detail in the reference)
- Incubation of 10 ml aliquots in artifical continuous light
- Cell multiplication measured turbidimetrically at 578 nm with an optical path length of 10 mm

Duration:

8 d

Dose descriptor:

other: toxicity threshold (TT, EC3)

Effect conc.:

3.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

TT (Toxicity Threshold) comparable to EC3

Validity criteria fulfilled:

not specified

Remarks:

Documentation is insufficient for assessment. It is unclear whether the algae are within the exponential growth throughout the whole exposure period of 8 days.

Executive summary:

For the cyanobacterium Microcystis aeruginosa in a cell growth inhibition test Bringmann (1975) reported a 8d-NOEC = 3.3 mg/L.

Reference 4

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Documentation is insufficient for assessment. It is unclear whether the algae are within the exponential growth throughout the whole exposure period of 8 days.

Principles of method if other than guideline:

other: Cell multiplication inhibition test according to Bringmann & Kuehn (1977)

GLP compliance:

not specified

Specific details on test material used for the study:

- Name of test material (as cited in publications): 4-Nitrotoluol
- Analytical purity: no data

Analytical monitoring:

no

Test organisms (species):

Scenedesmus quadricauda

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

8 d

Test temperature:

27 °C

pH:

7.0

Details on test conditions:

- 100 ml test solution (neutralized) of certain dilutions of the test substance prepared in replicate
- Test solution contains nutrient medium (all media for test and culture are described in detail in the reference)
- Incubation of 10 ml aliquots in Kapsenberg-culture vials in artifical continuous light
- Cell multiplication measured turbidimetrically at 578 nm with an optical path length of 10 mm

Duration:

8 d

Dose descriptor:

other: Toxicity Threshold (TT, EC3)

Effect conc.:

15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

TT (Toxicity Threshold) comparable to EC3

Validity criteria fulfilled:

not specified

Remarks:

It is unclear whether the algae are within the exponential growth throughout the whole exposure period of 8 days.

Executive summary:

The cell growth inhibition test with the unicellular green alga Scenedesmus quadricauda gave the following results (Bringmann and Kuehn, 1977): TTC (= EC3) 15 mg/L after 8 days.

Reference 5

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

other: OECD SIDS

Adequacy of study:

weight of evidence

Reliability:

other: OECD SIDS

Rationale for reliability incl. deficiencies:

other: no reliability is given as this is a summary entry for the OECD SIDS

Principles of method if other than guideline:

other: OECD SIDS

GLP compliance:

not specified

Test organisms (species):

other: OECD SIDS

Remarks on result:

other: key results see 'executive summary'

OECD SIDS (2003):

 

Several studies on the toxicity of 4-nitrotoluene to algae were summarized in the assessment of the OECD SIDS (2003).

For the algae Chlorella pyrenoidosa a 96 h-EC50 on a decrease of 50 % in the maximum density (yield) is reported with a nominal concentration of 22.2 mg/l (Deneer et al., 1988; Deneer et al., 1989). The test was conducted according to the modified OECD Guideline 201. Studies according to OECD Guideline 201 (except for the shorter exposure duration) on Scenedesmus obliquus yielded a 48 h-EC50 for growth rate inhibition of ca. 25 mg/l (nominal value) (Liu and Lang, 1995; Liu and Lang, 2000; Zhao et al., 1997; Lu et al., 2001).

In a toxicity test with the algae Scenedesmus pannonicus a 96 h-NOEC of 10 mg/l was observed for the endpoint biomass (nominal concentration) (Slooff and Canton, 1983).

Executive summary:

OECD SIDS (2003):

 

Several studies on the toxicity of 4-nitrotoluene to algae were summarized in the assessment of the OECD SIDS (2003).

For the algae Chlorella pyrenoidosa a 96 h-EC50 on a decrease of 50 % in the maximum density (yield) is reported with a nominal concentration of 22.2 mg/l (Deneer et al., 1988; Deneer et al., 1989). The test was conducted according to the modified OECD Guideline 201. Studies according to OECD Guideline 201 (except for the shorter exposure duration) on Scenedesmus obliquus yielded a 48 h-EC50 for growth rate inhibition of ca. 25 mg/l (nominal value) (Liu and Lang, 1995; Liu and Lang, 2000; Zhao et al., 1997; Lu et al., 2001).

In a toxicity test with the algae Scenedesmus pannonicus a 96 h-NOEC of 10 mg/l was observed for the endpoint biomass (nominal concentration) (Slooff and Canton, 1983).

Reference 6

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

other: BUA report

Adequacy of study:

weight of evidence

Reliability:

other: BUA report

Rationale for reliability incl. deficiencies:

other: no reliability is given as this is a summary entry for the BUA report

Principles of method if other than guideline:

other: BUA report

GLP compliance:

not specified

Test organisms (species):

other: BUA report

Remarks on result:

other: key results see 'executive summary'

BUA report (1989):

 

The cell growth inhibition test with the unicellular green alga Scenedesmus quadricauda gave the following results (Bringmann and Kuehn, 1977): TTC (= EC3) 15 mg/L after 8 days.

In a 96h-test with p-nitrotoluene Slooff & Canton (1983) found 10 mg/L to be the highest concentration without an inhibitory effect on the cell growth of the green alga Scenedesmus pannonicus.

Another study on the unicellular green alga Chlorella pyrenoidosa determined the mononitrotoluene concentrations which lead to a 50% reduction of the cell multiplication rate, compared to that of the controls, after 96 hours (Deneer et al., 1988): EC50= 22.2 mg/L after 96 hours.

For the cyanobacterium Microcystis aeruginosa in a cell growth inhibition test Bringmann (1975) reported a 8d-NOEC = 3.3 mg/L. Slooff & Canton (1983) found no growth inhibition of Microcystis aeruginosa by p-nitrotoluene at concentrations up to 3.2 mg/L in a 4-day test.

Validity criteria fulfilled:

not specified

Executive summary:

BUA report (1989):

 

The cell growth inhibition test with the unicellular green alga Scenedesmus quadricauda gave the following results (Bringmann and Kuehn, 1977): TTC (= EC3) 15 mg/L after 8 days.

In a 96h-test with p-nitrotoluene Slooff & Canton (1983) found 10 mg/L to be the highest concentration without an inhibitory effect on the cell growth of the green alga Scenedesmus pannonicus.

Another study on the unicellular green alga Chlorella pyrenoidosa determined the mononitrotoluene concentrations which lead to a 50% reduction of the cell multiplication rate, compared to that of the controls, after 96 hours (Deneer et al., 1988): EC50= 22.2 mg/L after 96 hours.

For the cyanobacterium Microcystis aeruginosa in a cell growth inhibition test Bringmann (1975) reported a 8d-NOEC = 3.3 mg/L. Slooff & Canton (1983) found no growth inhibition of Microcystis aeruginosa by p-nitrotoluene at concentrations up to 3.2 mg/L in a 4-day test.

Reference 7

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: 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)

Principles of method if other than guideline:

other: no guideline followed, see test conditions

GLP compliance:

no

Specific details on test material used for the study:

Chemicals were purchased from different commercial sources and were not repurified before testing.

Analytical monitoring:

no

Vehicle:

yes

Details on test solutions:

- Stock solutions of chemicals were prepared in acetone

Test organisms (species):

other: other algae: Scenedesmus obliquus

Test type:

not specified

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

24 +/- 1 °C

Details on test conditions:

- Algae were cultured in an unspecified growth medium
- The photoperiod was 12 hours under cool white fluorescent light of 4000 +/- 400 Lux, followed by 12 hours darkness
- 50 mL incubation solution in 100 ml sterile-closed flasks
- Initial algae density 10000 cells
- 5 different concentrations and controls, each 3 replicates
- Cell density determination after 0, 24, 48, 72, and 96 hours, optical density after 96 hours at 650 nm

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

ca. 25 mg/L

Nominal / measured:

not specified

Conc. based on:

not specified

Basis for effect:

growth rate

Validity criteria fulfilled:

not specified

Executive summary:

A test on the acute toxicity of 4-nitrotoluene to the alga Scenedesmus obliquus was performed. For a test period of 96 hours an EC50 value of ca. 25 mg/l was reported (Zhao, 1997).

Reference 8

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

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)

Principles of method if other than guideline:

other: Method according to Altenburger (1990)

GLP compliance:

not specified

Specific details on test material used for the study:

- Name of test material (as cited in publication): 4-Me-1-NO2
- purity > 97 %

Analytical monitoring:

not specified

Test organisms (species):

other: other algae: Scenedesmus vacuolatus

Details on test organisms:

- Scenedesmus vacuolatus was formerly referred to as Chlorella fusca
- Synchronization of the cells was imposed prior to testing by a 14h-10h light-dark change and a periodic dilution to the standard cell density.

Test type:

not specified

Water media type:

freshwater

Total exposure duration:

24 h

Test temperature:

28 °C +/- 0.5 °C

pH:

6.7

Details on test conditions:

- standard, initial cell concentration was approx. 1E5 cells/mL
- photoautotrophic growth of the cells was performed in gastight test vessels

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

17.2 mg/L

Nominal / measured:

not specified

Conc. based on:

not specified

Basis for effect:

growth rate

Details on results:

Result in original reference given as logEC50 (mol/L)

Validity criteria fulfilled:

not specified

Executive summary:

A test on the acute toxicity of 4-nitrotoluene to the alga Scenedesmus vacuolatus was performed according to the method reported by Altenburger (1990). For a test period of 24 hours an EC50 value of 17.2 mg/l was reported (Schmitt, 2000).

Reference 9

Endpoint:

toxicity to aquatic algae and cyanobacteria

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 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

: slight modifications according to van Leeuwen et al. (1985)

GLP compliance:

not specified

Specific details on test material used for the study:

- Name of test material (as cited in study report): 4-nitrotoluene
- purity: > 98 %

Analytical monitoring:

no

Test organisms (species):

Chlorella pyrenoidosa

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

CaCO3: 90 mg/L

Test temperature:

20 +/- 1 °C

pH:

8.7 +/- 0.2

Details on test conditions:

- initial cell density of approximately 10^8 cells/L
- 100 mL were added to 200 mL Erlenmeyer flasks,
which were stoppered with a cotton plug and
placed on a mechanical shaker under fluorescent light (7.5 Wm^-2)
- cells were counted in a model ZBI Coulter Counter, using a 70 µm aperture
- tests were carried out in triplicate

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

22 mg/L

Nominal / measured:

not specified

Conc. based on:

not specified

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 15-31

Details on results:

The 96h-EC50 values for effects on the yield of C. pyrenoidosa populations were calculated according to Kooyman et al.(1981).

Validity criteria fulfilled:

not specified

Executive summary:

The toxicity of 4-nitrotoluene towards the algae Chlorella pyrenoidosa was tested according to the OECD Guideline 201 with slight modifications according to van Leeuwen et al. (1985). The EC50 (96 h) for growth inhibition was determined to be 22 mg/L (Maas-Diepeveen et al., 1986).

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.

 

Weight of evidence 

The toxicity of 4-nitrotoluene towards the algae Chlorella pyrenoidosa was tested according to the OECD Guideline 201 with slight modifications according to van Leeuwen et al. (1985). The EC50 (96 h) for growth inhibition was determined to be 22 mg/L (Maas-Diepeveen et al., 1986).

 

OECD SIDS (2003):

Several studies on the toxicity of 4-nitrotoluene to algae were summarized in the assessment of the OECD SIDS (2003).

For the algae Chlorella pyrenoidosa a 96 h-EC50 on a decrease of 50 % in the maximum density (yield) is reported with a nominal concentration of 22.2 mg/l (Deneer et al., 1988; Deneer et al., 1989). The test was conducted according to the modified OECD Guideline 201. Studies according to OECD Guideline 201 (except for the shorter exposure duration) on Scenedesmus obliquus yielded a 48 h-EC50 for growth rate inhibition of ca. 25 mg/l (nominal value) (Liu and Lang, 1995; Liu and Lang, 2000; Zhao et al., 1997; Lu et al., 2001).

In a toxicity test with the algae Scenedesmus pannonicus a 96 h-NOEC of 10 mg/l was observed for the endpoint biomass (nominal concentration) (Slooff and Canton, 1983).

 

BUA report (1989):

The cell growth inhibition test with the unicellular green alga Scenedesmus quadricauda gave the following results (Bringmann and Kuehn, 1977): TTC (= EC3) 15 mg/L after 8 days.

In a 96h-test with p-nitrotoluene Slooff & Canton (1983) found 10 mg/L to be the highest concentration without an inhibitory effect on the cell growth of the green alga Scenedesmus pannonicus.

Another study on the unicellular green alga Chlorella pyrenoidosa determined the mononitrotoluene concentrations which lead to a 50% reduction of the cell multiplication rate, compared to that of the controls, after 96 hours (Deneer et al., 1988): EC50= 22.2 mg/L after 96 hours.

For the cyanobacterium Microcystis aeruginosa in a cell growth inhibition test Bringmann (1975) reported a 8d-NOEC = 3.3 mg/L. Slooff & Canton (1983) found no growth inhibition of Microcystis aeruginosa by p-nitrotoluene at concentrations up to 3.2 mg/L in a 4-day test.

A test on the acute toxicity of 4-nitrotoluene to the alga Scenedesmus vacuolatus was performed according to the method reported by Altenburger (1990). For a test period of 24 hours an EC50 value of 17.2 mg/l was reported (Schmitt, 2000).

A test on the acute toxicity of 4-nitrotoluene to the alga Scenedesmus obliquus was performed. For a test period of 96 hours an EC50 value of ca. 25 mg/l was reported (Zhao, 1997).

 

Conclusion
The EC50 and NOEC values obtained from these studies are in the same order of magnitude.

96h-EC50: 22.2 mg/l - 25 mg/L
96h-NOEC: 10 mg/L

The most conservative 96h-LC50 of 22.2 mg/l (Deneer et al., 1988; Deneer et al., 1989) and 96h-NOEC value of 10 mg/L (Slooff & Canton, 1983) is chosen as key value for assessment.

Key value for chemical safety assessment

EC50 for freshwater algae:

22.2 mg/L

EC10 or NOEC for freshwater algae:

10 mg/L