4-[(2,5-dichlorophenyl)azo]-3-hydroxy-N-(2-methoxyphenyl)naphthalene-2-carboxamide

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:

2010-01-08 to 2010-01-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study meets requirements of OECD Guideline 211 and GLP requirements with no deviations.

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-N-(o-tolyl)-4-[(2,4,5-trichlorophenyl)azo]naphthalene-2-carboxamide

EC Number:

229-440-3

EC Name:

3-hydroxy-N-(o-tolyl)-4-[(2,4,5-trichlorophenyl)azo]naphthalene-2-carboxamide

Cas Number:

6535-46-2

Molecular formula:

C24H16Cl3N3O2

IUPAC Name:

3-hydroxy-N-(2-methylphenyl)-4-[(2,4,5-trichlorophenyl)diazenyl]-2-naphthamide

Test material form:

solid: nanoform, no surface treatment

Details on test material:

Name of test material (as cited in study report): (no report title): Pigment Red 112
Analytical purity: 99.8 %

Sampling and analysis

Analytical monitoring:

no

Details on sampling:

No analytical determination of the test item was carried out due to the low water solubility of the test item.

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 1 mg/L prepared in dilution water was shaken with 20 rpm for 48 h at ambient temperature and filtrated with 0.45 µm RC filter.
- Eluate: Dilution water
- Differential loading: The saturated solution was tested as limit concentration.
- 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): 3 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:

The test medium had a nominal hardness of 0.24 mmol CaMg/L

Test temperature:

Mean: 21.5 °C, Min: 21 °C, Max: 22 °C

pH:

Nominal test item concentration pH-value
[mg/L] Start; 0 h End; 72 h
Saturated solution (1 mg/L) 8.04 8.35
Control 8.13 8.34

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

See above

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 636336 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
- Spacing factor for test concentrations: None, limit test
- Range finding study:
Results of the Range Finding Test (0-72 h)

Saturated solution [mg/L] Specific Growth Rate Inhibition [%] Yield Inhibition [%]
Filtration of the saturated solution
20 - 3 - 19
1 - 3 - 19

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1 other: mg/L (saturated solution)

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield

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
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50:
Rate-related inhibition: 0.60 (CI 0.545 - 0.653) mg/L
Yield Inhibition: 0.28 (CI 0.266 - 0.299) mg/L

Reported statistics and error estimates:

The NOEC and LOEC were determined by calculation of
statistical analyses 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 and 95 % Confidence Intervals of the test item (0-72 h)

                   based on nominal test item concentration [mg/L]

                               

	Rate-related Inhibition
NOEC	Saturated solution (1 mg/L)
LOEC	> Saturated solution (1 mg/L)
ErC10	> Saturated solution (1 mg/L)
ErC20	> Saturated solution (1 mg/L)
ErC50	> Saturated solution (1 mg/L)
	Inhibition of Yield
NOEC	Saturated solution (1 mg/L)
LOEC	> Saturated solution (1 mg/L)
EyC10	> Saturated solution (1 mg/L)
EyC20	> Saturated solution (1 mg/L)
EyC50	> Saturated solution (1 mg/L)

Cell Densities

	Nominal test item concentration		Replicate		Cell density [cells/mL]
[mg/L]		No.		0 h		24 h	48 h	72 h
Saturated solution              (1 mg/L)		1		5074		19924	172088	811286
		2		5074		22729	170534	778264
		3		5074		24729	147336	721561
		4		5074		22023	140071	772979
		5		5074		23323	172006	761032
		6		5074		21941	177028	809346
		Mean		5074		22445	163177	775745
Control		1		5074		22589	146618	743013
		2		5074		22778	126409	607983
		3		5074		21527	162481	677384
		4		5074		19227	151563	600533
		5		5074		25234	125864	562982
		6		5074		23782	161637	626122
		Mean		5074		22523	145762	636336

Evaluation after 72 h

              Statistically significant differences of growth rates and yield compared to

                   control values are marked (+), not significant differences are marked (-).

                              

Nominal test item concentration	Replicate	Growth rate		Rate-related inhibition	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
Saturated solution              (1 mg/L)	1		1.69	-5.12		806212	-27.7
	2		1.68	-4.26		773190	-22.5
	3		1.65	-2.69		716487	-13.5
	4		1.68	-4.11		767905	-21.7
	5		1.67	-3.79		755958	-19.8
	6		1.69	-5.07		804272	-27.4
	Mean	(+)*	1.68	-4.17	(+)*	770671	-22.1
Control	1		1.66			737939
	2		1.60			602909
	3		1.63			672310
	4		1.59			595459
	5		1.57			557908
	6		1.61			621048
	Mean		1.61			631262

Section-by-Section and Average Specific Growth Rates of the Control Group
               (0 – 72 h)

	Replicate No.	Specific growth rate [d^-1]			Mean (0 - 72 h)	SD ±	CV [%]	Mean CV [%]
		section-by-section
		0 - 24 h	24 - 48 h	48 - 72 h
Control	1	1.49	1.87	1.62	1.66	0.192	11.5	14.4
	2	1.50	1.71	1.57	1.60	0.108	6.78
	3	1.45	2.02	1.43	1.63	0.338	20.7
	4	1.33	2.07	1.38	1.59	0.411	25.8
	5	1.60	1.61	1.50	1.57	0.062	3.95
	6	1.55	1.92	1.35	1.61	0.286	17.8
				Mean	1.61
				± SD	0.03
				CV [%]	2.04

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study the test item was found not to inhibit the freshwater green algae Desmodesmus subspicatus after 72 h at the saturated solution (prepared with 1 mg/L). All effect levels are given based on the nominal concentration of Pigment Red 112.

Executive summary:

The toxicity of Pigment Red 112 (batch number 2468) to the unicellular freshwater green algae Desmodesmus subspicatus was determined according to the principles of OECD 201 at Dr.U.Noack-Laboratorien in 31157 Sarstedt, Germany from January 08 to 14, 2010 with the definitive exposure phase from January 11 to 14, 2010. The aim of the study was to assess the effects on growth rate and yield over a period of 72 h.

The study was conducted under static conditions with an initial cell density of approximately  2 – 5 x 10³cells/mL. Based on a preliminary range finding test, a limit test with a saturated solution*  was carried out. The saturated solution was prepared out of the dispersion of 1 mg/L. Six replicates were tested for the limit concentration and control. Environmental conditions were determined to be within the acceptable limits.

All effect values are given based on the nominal concentration of the test item.

NOEC, LOEC and EC - values and 95 % Confidence Intervals of the tets item (0-72 h)

                   based on nominal test item concentration [mg/L]

                               

	Rate-related Inhibition
NOEC	Saturated solution (1 mg/L)
LOEC	> Saturated solution (1 mg/L)
ErC10	> Saturated solution (1 mg/L)
ErC20	> Saturated solution (1 mg/L)
ErC50	> Saturated solution (1 mg/L)
	Inhibition of Yield
NOEC	Saturated solution (1 mg/L)
LOEC	> Saturated solution (1 mg/L)
EyC10	> Saturated solution (1 mg/L)
EyC20	> Saturated solution (1 mg/L)
EyC50	> Saturated solution (1 mg/L)