3-hydroxy-2'-methyl-2-naphthanilide

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-11-06 to 2009-11-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: study performed according to current guideline and GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

3-hydroxy-2'-methyl-2-naphthanilide

EC Number:

205-205-0

EC Name:

3-hydroxy-2'-methyl-2-naphthanilide

Cas Number:

135-61-5

Molecular formula:

C18H15NO2

IUPAC Name:

3-hydroxy-N-(2-methylphenyl)naphthalene-2-carboxamide

Constituent 2

Reference substance name:

Naphtol AS-D

IUPAC Name:

Naphtol AS-D

Details on test material:

- Name of test material (as cited in study report): Naphtol AS-D
- Molecular formula: C18H15NO2
- Molecular weight: 277.33 g/mol
- Physical state: Solid, beige powder

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The limit concentration and the control were analytically verified at the beginning and the end of the test via HPLC-DAD analysis.
- Sampling method: Samples at test start and end were taken directly from the test replicates.
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C until start of analysis, if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A saturated solution* was prepared. A dispersion of 100 mg/L prepared in dilution water was shaken with 20 rpm for 48 h at ambient temperature and centrifuged at 10000 rpm for 10 min.
- Eluate: Dilution water
- Differential loading: The saturated solution was tested in a limit test corresponding to a
geometric mean measured concentration of 0.711 mg/L.
- Controls: Six replicates without test item were tested under the same test conditions as the test replicates.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus Chodat
- Strain: SAG 86.81
- Source (laboratory, culture collection): SAG Pflanzenphysiologisches Institut der Universitaet Goettingen, Nikolausberger Weg 18, D-37073 Goettingen, Germany
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: Fresh stocks were prepared from Z-Agar. Light intensity amounted 35 - 70 µE x m-2 x s-1 for 24 h per day.


ACCLIMATION
- Culturing media and conditions (same as test or not): Same as test
- Any deformed or abnormal cells observed: No

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Hardness:

Not measured

Test temperature:

Mean: 22 °C, Min: 21 °C, Max: 23 °C

pH:

Geometric mean measured test item concentration pH-value
[mg/L] Start; 0 h End; 72 h
0.711 8.02 8.53
Control 8.12 8.54

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Please refer to "any other information on materials and methods incl. tables"

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at appr. 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: 5074 cells/mL
- Control end cells density: Mean 378432 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): None


GROWTH MEDIUM
- Standard medium used: yes



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dilution water, composition acc. to the guideline
- Hardness: 0.24 mmol Ca+Mg/L
- pH-value: 8.1 +/- 0.2
- Culture medium: Nutrient Medium Z acc. to LÜTTGE et al. (1994)


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: 60 - 120 µE x m-2 x s-1



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] Fluorimeter, via Chlorophyll-a fluorescence (excitation at 436 nm, emission at 685 nm), cell concentrations were related to all cell density values acc. to a calibration curve.



TEST CONCENTRATIONS
- Range finding study: Yes
Results of the Range Finding Test (0 – 72 h)


Saturated solution [%] Specific Growth Rate Inhibition [%] Yield Inhibition [%]
100 - 6 - 34
10 - 5 - 27
1 - 2 - 13

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.711 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and yield inhibition

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50:
Rate-related inhibition: 0.600 (CI 0.545 - 0.653) mg/L
Yield Inhibition: 0.280 (CI 0.266 - 0.299) mg/L

Reported statistics and error estimates:

The NOEC and LOEC were determined by calculation of statistical significance of growth rates and yield.
t-test was carried out for the determination of statistically significant differences compared to control replicates. When running a t-test a Normality Test and an Equal Variance Test were done first. P-values for both Normality and Equal Variance Tests were 0.05. The a-value was a=0.05. Detection of statistically significant differences were considered negligible when inhibition values were ≤ 10 %. These findings were marked biological not significant.

Any other information on results incl. tables

NOEC, LOEC and EC-Values of Naphtol AS-D (0-72 h)

                                based on the geometric mean measured test item concentration [mg/L]

                               

EC-Values	Rate-related inhibition
NOEC	0.711
LOEC	> 0.711
ErC10	> 0.711
ErC20	> 0.711
ErC50	> 0.711
	Yield inhibition
NOEC	0.711
LOEC	> 0.711
EyC10	> 0.711
EyC20	> 0.711
EyC50	> 0.711

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study the effects of the test item Naphtol AS-D on the growth of the freshwater green alga Desmodesmus subspicatus were tested. No inhibiting effects on specific growth rate and yield were found in the limit concentration of 0.711 mg/L. The EC50-values for specific growth rate inhibition (ErC50) and inhibition of yield (EyC50) after 72 h were > 0.711 mg/L.
All effect levels are given based on the geometric mean measured test item concentration.

Executive summary:

The toxicity of Naphtol AS-D (Batch no.: Changshu Huayi, Batch 040 267) to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201.

The concentration of Naphtol AS-D was determined at test start and end via HPLC-DAD analysis. The measured concentration at test end was 41 % of the initially measured test item concentration. All effect levels are given based on the geometric mean measured test item concentration.

NOEC, LOEC and EC-Values of Naphtol AS-D (0-72 h)

                                based on the geometric mean measured test item concentration [mg/L]

                               

EC-Values	Rate-related inhibition
NOEC	0.711
LOEC	> 0.711
ErC10	> 0.711
ErC20	> 0.711
ErC50	> 0.711
	Yield inhibition
NOEC	0.711
LOEC	> 0.711
EyC10	> 0.711
EyC20	> 0.711
EyC50	> 0.711