Diphenylamine

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:

key study

Study period:

June 1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Vehicle:

not specified

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Daphnids were exposed to a dilution series of the substances prepared in IS0 test medium (IS0 1982) based on deionised water. The tests were run in 30 ml plastic vessels each containing 10 ml of test solution.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnid
- Source: Lake Langedam, Denmark
- Age at study initiation (mean and range, SD): less than 24 h old

Test type:

not specified

Water media type:

not specified

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

after the exposure period

Hardness:

Concerning hardness, no information is clearly given inthe document, consequently we consider that the estimation is done for 10-250 mg CaCO3/L.

Test temperature:

Concerning temperature, no information is clearly given inthe document, consequently we consider that the estimation is done for relevant natural environmental conditions (20°C).

pH:

Concerning pH, no information is clearly given in the document, consequently we consider that the estimation is done for 6.0-8.5 .

Dissolved oxygen:

Concerning dissolved oxygen, no information is clearly given inthe document, consequently we consider that the estimation is done for at least 60% of the air saturation.

Salinity:

freshwater test

Nominal and measured concentrations:

no data

Details on test conditions:

TEST SYSTEM
- Test vessel: yes
- Material, size, headspace, fill volume: 30 ml plastic vessels containing 10 ml of test substance
- No. of organisms per vessel: 5 animals
- No. of vessels per concentration (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO test medium based on deionised water

Reference substance (positive control):

not specified

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

2 mg/L

Conc. based on:

other: as NVOC in lowest, medium and highest concentrations

Basis for effect:

mobility

Remarks on result:

other: 95 % CL: 1.69-2.46 mg/L

Results with reference substance (positive control):

no data

Reported statistics and error estimates:

no data

Applying the EU classification criteria for environmental danger, the following classification of diphenylamine can be proposed: N: R50/53, i.e. "Very toxic to aquatic organisms and may cause long-term effects in the aquatic environment"

Validity criteria fulfilled:

not specified

Remarks:

details of the results not available

Conclusions:

The 48 h-EC50 for immobilisation of Daphnia magna was estimated to be 2.0 mg/L: diphenylamine is very toxic to the tested species

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

ISO 6341 (Water quality - Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea))

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: Swedish Standard SS 02 8106 (Determination of acute lethal toxicity of chemicals substances and effluents to Nitocra spinipes)

Deviations:

not specified

GLP compliance:

not specified

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
PHYSICO-CHEMICAL PROPERTIES
- Melting point: 53°C
- Boiling point: 302°C
- Vapour pressure: 0.0033 Torr at 20°C
- Water solubility (under test conditions): 52±54 mg/l at 20°C
- log Pow: 3.62

Analytical monitoring:

not specified

Vehicle:

yes

Test organisms (species):

other: Nitocra spinipes and Daphnia magna

Details on test organisms:

- Age at study initiation (mean and range, SD): about 2±4 weeks old

The daphnids (D. magna clone 1) were obtained from laboratory cultures and cultured as in ISO (1996). The culture medium was prepared from
well water and the daphnids in the cultures (but not during the bioassays) were fed daily by a synthetic diet.

Test type:

not specified

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

after the exposure period

Hardness:

Concerning hardness, no information is clearly given inthe document, consequently we consider that the estimation is done for 10-250 mg CaCO3/L.

Test temperature:

20 +/- 2°C

pH:

Concerning pH, no information is clearly given in the document, consequently we consider that the estimation is done for 6.0-8.5 .

Dissolved oxygen:

The measured values for dissolved oxygen (initial and final) were above 80% of air saturation in all tests.

Salinity:

Salinity during culture and testing was 7 psu (practical salinity units g/l)

Nominal and measured concentrations:

no data

Details on test conditions:

Toxicity testing of N. spinipes:

A concentration series was made by progressive dilution (factor, 0.5) with 7 psu (g/l) seawater. Exposures were made in 3-ml wells on disposable Nunc 24 well plates.
The control was made of 7 psu seawater, and a test series with the reference toxicant, potassium dichromate, was made in parallel with each test run. Clean sediment from the Faeroe Islands was added to one series at 10% wet wt. Prior to addition of test organisms, all test solutions and/or suspensions were allowed to settle for 1±2 h.
After addition of test organisms to each well, the plates were incubated in total darkness at 20 +/- 2°C.

TEST SYSTEM
- No. of organisms per vessel: 5

Nitocra are cultured and tested in darkness to standardise illumination intensity.
Survival was recorded by counting the number of live animals after 96 h, and the 96-h LC50 was determined.

Toxicity testing of D. magna:

The toxicity tests with D. magna were made according to ISO (1996) with or without addition of 10% (wet wt.) clean sediment in Petri dishes (i.d. 10 cm)with a volume of 50 ml.
Test solutions were prepared by diluting 16 g sediment to 100 ml with ISO (1996) standardised dilution water, mixing, distributing equal volumes into two Petri dishes. The solutions were allowed to settle for 1±2 h prior to addition of 20 new-born daphnids to each dish. Immobility was recorded after 24 and 48 h.
Temperature during exposure was 20 +/- 2°C and the photoperiod was 12 h light and 12 h darkness. Dilution water controls and a test series with potassium dichromate were made in parallel to each test run. The measured values for dissolved oxygen were above 80% of air saturation in all tests.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

48 h

Dose descriptor:

EC50

Remarks:

Daphnia

Effect conc.:

1.2 mg/L

Nominal / measured:

not specified

Conc. based on:

not specified

Basis for effect:

mobility

Remarks:

(in new water)

Remarks on result:

other: 0.14 mg/L + UV light

Duration:

48 h

Dose descriptor:

EC50

Remarks:

Daphnia

Effect conc.:

2.6 mg/L

Nominal / measured:

not specified

Conc. based on:

not specified

Basis for effect:

mobility

Remarks:

(in aged water)

Remarks on result:

other: 0.58 mg/L + UV light

Duration:

48 h

Dose descriptor:

EC50

Remarks:

Daphnia

Effect conc.:

3.2 mg/L

Nominal / measured:

not specified

Conc. based on:

not specified

Basis for effect:

mobility

Remarks:

(in new sediment)

Remarks on result:

other: 0.60 mg/L + UV light

Duration:

96 h

Dose descriptor:

EC50

Remarks:

Nitocra

Effect conc.:

3.7 mg/L

Nominal / measured:

not specified

Conc. based on:

not specified

Basis for effect:

mortality

Remarks:

(in new water)

Remarks on result:

other: 1.0 mg/L + UV light

Duration:

96 h

Dose descriptor:

EC50

Remarks:

Nitocra

Effect conc.:

> 11 mg/L

Nominal / measured:

not specified

Conc. based on:

not specified

Basis for effect:

mortality

Remarks:

(in aged water)

Remarks on result:

other: same result + UV light

Duration:

96 h

Dose descriptor:

EC50

Remarks:

Nitocra

Effect conc.:

1.22 mg/L

Nominal / measured:

not specified

Conc. based on:

not specified

Basis for effect:

mortality

Remarks:

(in new sediment)

Remarks on result:

other: 0.86 mg/L + UV light

Duration:

96 h

Dose descriptor:

EC50

Remarks:

Nitocra

Effect conc.:

> 10

Nominal / measured:

not specified

Conc. based on:

not specified

Basis for effect:

mortality

Remarks:

(in aged sediment)

Remarks on result:

other: same result + UV light

Reported statistics and error estimates:

Probit analysis or moving average according to Peltier and Weber (1985) was used for determination of 24- and 48-h EC50s for Daphnia and 96-h LC50s for Nitocra. Differences in toxicity (48-h EC50s for Daphnia and 96-h LC50s for Nitocra) due to chemical tested (picric acid, TNT, RDX, DNT and diphenylamine), test medium (water only and plus sediment), ageing (directly and after storage for 30 d), UV activation (illumination for 2 h after termination of regular test), and test species and salinity (Daphnia or Nitocra) was tested by analysis of variances (ANOVA) with the aid of a statistical software package (Crunch ver. 4, Crunch, Software Corp., Oakland, CA, USA).

The results for Daphnia from the aged sediment tests were not valid because of excessive mortality in the controls.

Validity criteria fulfilled:

yes

Remarks:

survival in controls > 90 %; good sensitivity to the reference substance toxicant potassium dichromate

Conclusions:

In this study, diphenylamine was the most toxic of all the chemicals tested
Both ageing and test organism significantly affect its toxicity: ageing in water (hydrolysis) was found to decrease toxicity, and Daphnia was more sensitive than Nitocra

Description of key information

EC50 48 hr (mobility): 2 mg/l for Daphnia magna (OECD 202)

EC50 48 hr (mobility in new and aged water): 1.2 mg/l and 2.6 mg/l for Daphnia magna (ISO 6341 15)

LC50 96 hr (mortality in new and aged water): 3.7 and > 11 mg/l for Nitocra spinipes (ISO 6341 15)

LC50 96 hr (mortality in new and aged sediment): 1.22 and > 10 mg/l for Nitocra spinipes (ISO 6341 15)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

1.2 mg/L

Marine water invertebrates

Marine water invertebrates

Effect concentration:

1.22 mg/L

Additional information

The key value of 1.2 mg/l for the EC50 48 hr for freshwater invertebrates was chosen regarding the highest reliability for the study and the lowest value for EC50 48 hr; this value was used for classification.

The key value of 1.22 mg/l for the LC50 96 hr for marine invertebrates was chosen regarding the lowest value of LC50 96 hr in the study of Dave & al. (2000). Thanks to this study, it can be considered that the toxicity of diphenylamine depends on the test species, the hydrolysis (new/aged water), biodegradation (new/aged sediment) and photoactivation of the substance.