N-1-naphthylaniline

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:

disregarded due to major methodological deficiencies

Study period:

26.09.1995 to 29.09.1995

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Relevant methodological deficiencies (number of replicates in the treatment group is not stated, probably only one; pH value deviated from the range given in the guideline; analytical determination of the test substance could not be achieved, below detection limit at test start and test end; inhibition of biomass production of 35% should have led to the conduct of a full study)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

yes

Remarks:

increase of pH of more than 1.5

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 1 mg/L
- Sampling method: no data
- Measurement of test concentration (1 mg/L), blank control and control without algae, immediately after test start and after 72 hours

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: To accelerate the dissolution process, 3 mg of the test substance were treated in an ultrasonic bath for 1 h and stirred for 24 h on a magnetic stirrer. Undissolved material was filtrated off, and only the dissolved portion was used for the test.
- Controls: yes, blank control (without test substance) and control without algae

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: Scenedesmus subspicatus CHODAT
- Source (laboratory, culture collection): Pflanzenphysiologisches Institut, University of Göttingen, Germany
- Method of cultivation: cultivation in the laboratory in an illuminated cabinet at 23 +/- 2°C, photosyntheticaly active irradiation of 120 µE/m²s, weekly inoculation of stem cultures

ACCLIMATION
- Culturing media and conditions (same as test or not): yes

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

no data

Test temperature:

no data

pH:

control: 8.3 (0 h), 10.4 (72 h)
1 mg/L: 8.4 (0 h), 10.4 (72 h)

Dissolved oxygen:

no data

Nominal and measured concentrations:

nominal: 0 (both controls); 1 mg/L
measured: < 0.5 mg/L*; < 0.5 mg/L* (at test start and after 72 h, respectively)

* detection limit

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type: closed
- Material, size, headspace, fill volume: 300 mL Erlenmeyer flasks with ground-in stoppers, 100 mL test medium
- Aeration: no data
- Initial cells density: 10,000 cells/mL (prepared from exponentially growing preculture)
- Control end cells density: 347,000 cells/mL
- No. of organisms per vessel: 10,000 cells/mL
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionized water, treated in Millipore system
- Intervals of water quality measurement: no dat

OTHER TEST CONDITIONS
- Photoperiod: incubation in illuminated cabinet with shaking device

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations: electronic particle counter (Microcellcounter Sysmex F-30 Digitana), daily determination

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

>= 0.25 mg/L

Basis for effect:

growth rate

Remarks on result:

other: EC50 determined as half of detection limit

Details on results:

- Exponential growth in the control (for algal test): yes
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values.

Reported statistics and error estimates:

Multiple comparison of the means using Dunnett's test against the control (Easy Assay Multiple Testing, Version 3.1)

Cell numbers [cells/mL]:

control:       76,700 after 24 h; 227,000 after 48 h; 347,000 after 72 h

1 mg/L         35,600 after 24 h; 132,000 after 48 h; 302,000 after 72 h

Biomass integral (over time):

control:       452,000

1 mg/L       294,000; corresponding to 35% inhibition of cell increase

Growth rate [1/d]:

control:      1.2

1 mg/L:      1.1; corresponding to 8.3% inhibition of growth   

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study following national guideline (of South Korea)

Qualifier:

according to guideline

Guideline:

other: Growth inhibition study in algae of Notification No. 1997-9 of NIER adopted in 1997

GLP compliance:

not specified

Analytical monitoring:

no

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: pre-test: 100 mg of the test substance was placed into a 10 mL flask with a drop of HCO-40 and DMSO added up to the demarcated line to prepare a 10 g/L stock solution. The stock solution was diluted with DMSO to produce stock solutions for each exposed concentration. These were used for the preparation of the test concentrations by sterilizing them after passing them through Acrodisc 0.2 µm for organic solvent and putting them in an agar medium of 10 mL volume.
main test: 64 mg of the test substance was placed into a 10 mL flask with a drop of HCO-40 and DMSO added up to the demarcated line to prepare a 6.4 g/L stock solution. The stock solution was diluted with DMSO to produce stock solutions for each exposed concentration. These were used for the preparation of the test concentrations by sterilizing them after passing them through Acrodisc 0.2 µm for organic solvent and putting them in an agar medium of 100 mL volume.
- Controls: only agar medium was used for the control group. For the solvent group a drop of HCO-40 was put into a flask of 10 mL volume and filled with DMSO up to the demarcated line. Then it was sterilized by putting it through Acrodisc 0.2 µm for organic solvent.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): HCO-40, DMSO

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: ATCC 22662
- Source (laboratory, culture collection): American Type Culture Collection, Rockville, Maryland, US)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

22.7 - 25.1 °C

pH:

7.8 - 7.9 (test start)
7.5 - 9.7 (test end)

Nominal and measured concentrations:

Nominal: 0.4, 0.8, 1.6, 3.2 and 6.4 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 150 mL Erlenmeyer flask containing 100 mL test solution
- Initial cells density: 10E4 cells/mL
- Control end cells density: 4.3E6 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- No. of vessels per vehicle control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: agar medium
- Culture medium different from test medium: no
- Intervals of water quality measurement: temperature was consecutively recorded during the study. pH was measured at the start and at the end of the test in a pH measuring flask.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: continuous light
- Light intensity and quality: 7796 - 8022 lux
- other: The vessels were shaken at 100 rpm throughout the test.

EFFECT PARAMETERS MEASURED: Algal cells were counted daily
- Determination of cell concentrations: Thomas hematocytometer

TEST CONCENTRATIONS
- Range finding study: yes
- Test concentrations: 0.1, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study:

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

0.93 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 0.92 - 0.95 mg/L

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

0.34 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 0.31 - 0.37 mg/L

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Turbidity observed in th 6.4 mg/L treatment.

Validity criteria fulfilled:

yes

Conclusions:

The test substance showed to be very toxic to green algae in a bioassay screening the toxicity (96h) of the test substance.

Description of key information

EC50(96h) = 0.93 mg/L (nominal, vehicle) for Pseudokirchneriella subcapitata (national korean guideline)

Key value for chemical safety assessment

EC50 for freshwater algae:

0.93 mg/L

Additional information

One reliable study investigating the toxicity of N-phenyl-1-naphthylamine to aquatic algae is available. The study was performed by the South Korean Ministry of Environment under staic conditions according to a national korean guideline (Yook 1998). HCO-40 and DMSO were used as vehicle to prepare test concentrations between 0.4 and 6.4 mg/L. The test substance was exposed to Pseudokirchneriella subcapitata over a test period of 96 hours. The 96h-EC50 value is determined to be 0.93 mg/L.

In addition, two futher studies are available, which, however, are disregarded for the hazard assessment of the substance. The first study investigated the effect of Vulkanox PAN (Additin 30) on growth rate and biomass production of Scenedesmus subspicatus in a GLP study according to EU method C.3 (Caspers 1996). One nominal concentration of 1 mg/L was tested. This study yielded an EC50 for growth rate inhibition of >= 0.25 mg/L. However, this study suffers from several relevant methodological deficiencies. The number of replicates in the treatment group is not stated (probably only one) and the pH value deviated from the range given in the guideline. Besides, an analytical determination of the test substance could not be achieved, the measured concentrations were below the detection limit at test start and test end. Finally, the inhibition of biomass production of 35% should have led to the conduct of a full study which was not carried out. Therefore, this study was not considered for the hazard assessment of the substance. The second study was obtained in the J-CHECk database of the Japanese Ministry of Environment. The few details of this study are given in Japanese. Translation of the available data indicate that a 72h algal toxicity test was performed. However, many important information about the test material, test design, reliability of the results as well as accuracy of the test performance are missing. Thus the reliability of the test results (particularly of importance since the results differ from that of a reliable study) cannot be evaluated. Consequently the data were not used for the hazard and risk assessment of the substance.