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

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-01-04 to 2013-01-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed according to relevant guidelines and compliant to GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP according to German Chemikaliengesetzt and Directive 88/320/EEC

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 1 mg/L, freshly prepared with dilution water. The stock solution was stirred for 48 hours (1100 rpm) with a magnetic stirrer. Undissolved particles were removed by membrane filtration (membrane filter 0.45 µm, RC, MACHEREY-NAGEL).

- Eluate: Dilution water
- Differential loading: Saturated solution* corresponding to a nominal test item concentration of 1.00 mg/L.

- Controls: Six replicates without test item were tested under the same test conditions as the test replicates.

*) Saturated solution: The maximum dissolved concentration of the test item that can be achieved under the test conditions in the test medium, acc. to OECD Series, No. 23 (2000)6.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus CHODAT SAG 86.81
- 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): Not the same, standard growth medium
- Any deformed or abnormal cells observed: No

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

None

Hardness:

Not measured; the synthetic medium used as dilution water had a nominal hardness of 0.24 mmol Ca+Mg/L

Test temperature:

Mean: 22.5 °C, Min: 22.0 °C, Max: 23.0 °C

pH:

Nominal concentration pH-value
[mg/L] Start; 0 hours End; 72 hours
1.00 7.67 8.29
Control 7.91 8.47

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Nominal limit concentration of test item used to prepare saturated solutions: 1 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): sealed with cotton wool plugs
- 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: 4332 cells/mL
- Control end cells density: Mean 944265 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:
According to the guideline (OECD)

Composition of Dilution water (OECD)

Component Concentration [mg/L]
NH4Cl 15
MgCl2 ¿ 6 H2O 12
CaCl2 ¿ 2 H2O 18
MgSO4 ¿ 7 H2O 15
KH2PO4 1.6
FeCl3 ¿ 6 H2O 0.064
Na2EDTA ¿ 2 H2O 0.1
H3BO3 0.185
MnCl2 ¿ 4 H2O 0.415
ZnCl2 3 x 10-3
Na2MoO4 ¿ 2 H2O 7 x 10-3
CoCl2 ¿ 6 H2O 1.5 x 10-3
CuCl2 ¿ 2 H2O 1 x 10-5
NaHCO3 50
pH-value 8.1 ¿ 0.2
This medium had a nominal hardness of 0.24 mmol Ca+Mg/L.


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)

Nominal Test Item Concentration [mg/L] Growth Rate Inhibition [%] Yield Inhibition [%]
Saturated Solution 1% 8%

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

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: 0.631 (CI 0.583 - 0.687) mg/L
Yield Inhibition: 0.256 (CI 0.247 - 0.265) mg/L

Reported statistics and error estimates:

NOEC/LOEC was determined by calculation of statistical significance of growth rates and yield. As standard One Way Analysis of Variance (ANOVA) and DUNNETT’s Test were used for NOEC/LOEC calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. The SHAPIRO-WILK-Test was used to test for normally distributed populations. The Levene median test was used for equal variance. P-values for both Normality and Equal Variance test were 0.05. The a-value (acceptable probability of incorrectly concluding that there is a difference) is a=0.05.
EC-values of the growth rate and yield inhibition were estimated empirically based on the results of the only treatment level (Limit test design).

Detection of statistically significant differences was considered negligible when inhibition values were = 10 %. These findings were marked as “biologically not significant”. Following the recommendations of the UBA (HEGER ET AL. 1998), inhibitions > 10 % are biologically significant.

NOEC, LOEC, EC - values of test item (0-72 hours)

                   based on nominal concentration [mg/L]

	Growth Rate Inhibition
NOEC	1.00
LOEC	> 1.00
ErC10	> 1.00
ErC20	> 1.00
ErC50	> 1.00
	Inhibition of Yield
NOEC	1.00
LOEC	> 1.00
EyC10	> 1.00
EyC20	> 1.00
EyC50	> 1.00

Cell Densities

	Nominal concentration		Replicate		Cell density [cells/mL]
[mg/L]		No.		0 hours		24 hours	48 hours	72 hours
1.00		1		4332		20789	146674	1030721
		2		4332		22044	152279	1037420
		3		4332		19751	155084	1141677
		4		4332		22082	164079	1099880
		5		4332		20887	148630	1047767
		6		4332		20251	167016	1099597
		Mean		4332		20967	155627	1076177
Control		1		4332		23780	173092	980307
		2		4332		23774	162544	938132
		3		4332		23107	156763	936434
		4		4332		23104	157116	988798
		5		4332		19956	151159	998768
		6		4332		20428	160701	823151
		Mean		4332		22358	160229	944265

Evaluation after 72 hours

                   Statistically significant differences of growth rates and yield compared to

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

Nominal concentration	Replicate	Growth rate		Growth rate inhibition	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
1.00	1		1.82	-1.67		1026389	-9.20
	2		1.83	-1.79		1033088	-9.91
	3		1.86	-3.57		1137345	-21.0
	4		1.85	-2.87		1095548	-16.6
	5		1.83	-1.97		1043435	-11.0
	6		1.85	-2.87		1095265	-16.5
	Mean	(+)^1	1.84	-2.46	(+)^1	1071845	-14.0
Control	1		1.81			975975
	2		1.79			933800
	3		1.79			932102
	4		1.81			984466
	5		1.81			994436
	6		1.75			818819
	Mean		1.79			939933

negative inhibitions = increase of growth - 1) Due to stimulation of growth

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

	Replicate No.	Specific growth rate [d^-1]			Mean (0 - 72 h)	SD ±	CV [%]	Mean CV [%]
		section-by-section
		0 - 24 hours	24 - 48 hours	48 - 72 hours
Control	1	1.70	1.99	1.73	1.81	0.155	8.56	9.74
	2	1.70	1.92	1.75	1.79	0.115	6.42
	3	1.67	1.92	1.79	1.79	0.120	6.71
	4	1.67	1.92	1.84	1.81	0.124	6.86
	5	1.53	2.03	1.89	1.81	0.257	14. 2
	6	1.55	2.06	1.63	1.75	0.275	15.7
				Mean	1.80
				SD ±	0.02
				CV [%]	1.33

Validity criteria fulfilled:

yes

Conclusions:

In this study no inhibition of growth of the freshwater green alga Desmodesmus subspicatus was found after 72 hours exposure to the test item. The NOEC for inhibition of specific growth rate and yield after 72 hours was 1.00 mg/L.
The test item caused a slight stimulation of the algae growth. All effect levels are based on the nominal concentration of the test item.

Executive summary:

The toxicity of the test item to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of
OECD 201 compliant to GLP (reliability category 1). The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 4332 cells/mL. A saturated solution* with a nominal concentration of 1 mg/L was tested. Six replicates were tested for the limit concentration and the control. Environmental conditions were determined to be within the acceptable limits.

No analytical determination was carried out since no suitable method for determination of the test item could be established: The test item is highly insoluble in water. All effect values of the test item are given based on the nominal concentration.

*) Saturated solution: The maximum dissolved concentration of the test item that can be achieved under the test conditions in the test medium, acc. to OECD Series, No. 23 (2000)6.

NOEC, LOEC, EC - values of the test item (0-72 hours)

                   based on nominal concentration [mg/L]

	Growth Rate Inhibition
NOEC	1.00
LOEC	> 1.00
ErC10	> 1.00
ErC20	> 1.00
ErC50	> 1.00
	Inhibition of Yield
NOEC	1.00
LOEC	> 1.00
EyC10	> 1.00
EyC20	> 1.00
EyC50	> 1.00

Description of key information

Algae

PR002

In the algal study (OECD 201) no inhibition of growth of the freshwater green alga Desmodesmus subspicatus was found after 72 hours exposure to the test item. The NOEC for inhibition of specific growth rate and yield after 72 hours was 1.00 mg/L.
The test item caused a slight stimulation of the algae growth. All effect levels are based on the nominal concentration of the test item.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

1 mg/L