2,6-dichloro-N-phenylaniline

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

other information

Study period:

February 07 - March 01, 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented and reported study, conducted equivalent to internationally accepted technical guideline in recognized research organization.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

,1984

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2,6-dichloro-N-phenylaniline

EC Number:

239-349-0

EC Name:

2,6-dichloro-N-phenylaniline

Cas Number:

15307-93-4

Molecular formula:

C12H9Cl2N

IUPAC Name:

2,6-dichloro-N-phenylaniline

Details on test material:

Identification Code: PBS 3247
Generic/Trade name: Dichlordiphenylamin dest.
BatchNo.: EN 322165.36
Appearance: beige solid
Purity: not available*
Solubility (in water): 0.0063 g/l
Received: 17/10/94
Storage: room temperature
Stability : 12/95

*: The test substance is identified by the code number. Identification and characterization is guaranteed by the sponsor. Data for purity were not available or not generated and documented according to the rules of Good Laboratory Practices.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Composite samples (about 150 ml per concentration and control) of each test concentration were drawn by mixing identical volumes of the test solutions taken from the test vessels. SampIes of test solutions were taken immediately before exposure and after 72 hours exposure and stock solutions were taken 24 hours before exposure. All samples were kept at - 18°C to - 22°C until analysis.

Test solutions

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- (Test) Water: composition according to the guideline
- Method: Stock solution: 100.4 mg test substance were mixed with 1.0 ml of a 0.2% TWEEN 80 solution in water (w/w) by a rubbowl and this suspension was then made up to 1000 ml with water. Calculated amounts of the stock solution to produce the desired test concentrations and vehicle to achieve identical vehicle concentrations were given into the water and were homogeneously distributed. The test solutions were equilibrated for 24 hours under test conditions.
- Controls: blank (test water) and vehicle control
- Chemical name of vehicle: polyoxy-ethylene-sorbitan-monooleate (TWEEN 80)
- Concentration of vehicle in test medium: stock solution: 2 mg/l, final test solutions including vehicle control: 0.116 mg/l
- Evidence of undissolved material: at nominal test concentrations 3.2 and 5.8 mg/l a deposit and some test substance on the surface of the test water were observed.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Strain: 86.81 SAG
- Source (culture collection): Collection of algae cultures, Pflanzenphysiologisches Institut, University Nikolausberger Weg 18, 37073 Göttingen/Germany
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: according to the guideline cited

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions: same as test
- Any deformed or abnormal cells observed: not reported

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

not indicated; the composition of the test water was according to the guideline

Test temperature:

24 ± 1 °C

pH:

test start: 8.0
test end: 8.1 - 9.1

Dissolved oxygen:

not applicable

Salinity:

not applicable (freshwater)

Nominal and measured concentrations:

nominal concentration: 0.10, 0.18, 0.32, 0.58, 1.0, 1.8, 3.2, and 5.8 mg/l
measured at test start: 0.04, 0.09 ,0.19 ,0.28 ,0.41 ,0.99 ,0.98, and 2.97 mg/l
measured at test end: 0.04, 0.06, 0.12, 0.17, 0.31, 0.70, 1.28, and 2.02 mg/l
arithmetric mean: 0.04, 0.08, 0.16, 0.23, 0.36, 0.85, 1.13, and 2.50 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 ml Erlenmeyer flasks, cotton stoppers, 50 ml test solution per flask
- Type (delete if not applicable): open
- Aeration: agitation on a lab shaker
- Initial cells density: 10700 cells/ml
- Control end cells density:
blank: 2'029'000 cells/ml
vehicle: 1'676'000 cells/ml
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 6


GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: composition according to the guideline
- Culture medium different from test medium: no
- Intervals of water quality measurement: Temperature was continuously measured; pH was measured at 0h and 72h exposure


OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: Continuous illumination
- Light intensity and quality: cold white fluorescent light, 113 µE/m2sec (approx. 8000 lux)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : cell density after 24, 48 and 72 hours exposure
- Determination of cell concentrations: cell counter


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Range finding study: results not reported

Reference substance (positive control):

not required

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.62 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 0.58-0.66

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.23 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.28 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

(area under growth curves)

Remarks on result:

other: 95% CL: 0.27-0.30

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.08 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

(area under growth curves)

Details on results:

- Exponential growth in the control (for algal test): yes
- Any stimulation of growth found in any treatment: at nominal test concentrations of 0.04 and 0.09 mg/l, slight stimulation of growth was observed (5.6-7.5% stimulation for biomass and 1.4% for growth rate, respectively).

Reported statistics and error estimates:

The EC-50 values were calculated according to the maximun likelihood method, logit model (Mc Cullagh, P., NeIder, J.A., 1983: Generalized linear models, Chapman & Hall, London).
slope: 2.4 for ErC50, 3.8 for EbC50. NOEC values were calculated on a 5% level.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

Criteria of OECD Guideline 201 (1984): Increase of the cell concentrations in the control cultures by a factor of at least 16 within three days).

Conclusions:

72h-EC50: >0.1 ≤1 mg/l

Executive summary:

In a 96-h toxicity study, green algae (Scenedesmus subspicatus) were exposed to PBS 3247 in accordance with OECD Guideline 201(Freshwater Alga and Cyanobacteria, Growth Inhibition Test) of 1984. The study was performed in compliance with GLP. Reliability grade 1 was assigned. Nominal concentrations of 0 (control and solvent control), 0.10, 0.18, 0.32, 0.58, 1.0, 1.8, 3.2, and 5.8 mg/l were tested. The toxicity endpoints were calculated based on arithmetic mean concentrations of 0.04, 0.08, 0.16, 0.23, 0.36, 0.85, 1.13 and 2.50 mg/l:

Inhibtion (growth rates):
ErC50 (0 - 72 h):	0.62 mg/l
95 % confidence limit:	0.58-0.66
slope:	2.4
NOErC ( 0-72 h): (5 % level)	0.23 mg/I
Inhibition (areas under growth curves):
EbC50 (0 - 72 h):	0.28 mg/I
95 % confidence limit:	0.27-0.30
slope:	3.8
NOEbC (0-72 h): (5 % level)	0.08 mg/l