alkoxy substituted aryl diazo heterocyclic aryl nitrile

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:

2015-03-08 to 2015-03-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The saturated solution and further four dilution levels out of the saturated solution were tested in a geometrical series with a dilution factor of 2: 6.25 - 12.5 - 25.0 - 50.0 - 100 % of the saturated solution, corresponding to the nominal loading rates 0.313 - 0.625 - 1.25 - 2.50 - 5.00 mg/L. The loading levels are based on a proposal of the sponsor.


- Sampling method: The test concentrations of Disperse Yellow DYLA 1306 were analytically verified via HPLC-UV in the saturated solution and the control at the start of the exposure (0 hours). At the start of the exposure (0 hours), sampling was carried out after preparation of the loading levels.
As the measured test item concentration was below the LOQ in the saturated solution, the test item concentrations at the other loading levels were considered to be also below the LOQ. At the end of the exposure (96 hours), no analytical monitoring was carried out, because a measured test item concentration above the LOQ was not observed at the start of the exposure.
The method was validated prior to this study according to SANCO 3029/99 rev.4 (2000).

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The saturated solution with a nominal loading of 5.00 mg/L was prepared once with dilution water. The saturated solution was stirred for 24 hours (1100 rpm) at room temperature with a magnetic stirrer. After 24 hours undissolved particles were removed by membrane filtration (membrane filter 0.2 µm, RC, MACHEREY-NAGEL). The filtrate was clear and yellow-orange.


- Differential loading: The saturated solution and further four dilution levels out of the saturated solution were tested in a geometrical series with a dilution factor of 2: 6.25 - 12.5 - 25.0 - 50.0 - 100 % of the saturated solution, corresponding to the nominal loading rates 0.313 - 0.625 - 1.25 - 2.50 - 5.00 mg/L. The loading levels are based on a proposal of the sponsor.

- Dilution water: Threefold concentrated AAP medium according to the guidelines
-Controls: Eight replicates (without test item) were tested under the same test conditions as the test item groups.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata HINDÁK
- Strain: SAG 61.81
- 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 hours per day.

ACCLIMATION
- Culturing media and conditions (same as test or not): Not the same, Nutrient medium Z according to LÜTTGE et al. (1994)
- Any deformed or abnormal cells observed: No

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

None

Hardness:

Not measured

Test temperature:

Mean: 24.4 °C, Min: 23.8°C, Max: 25.0 °C

pH:

Nominal test item
loading rate pH-value
[mg/L] Start; 0 hours End; 96 hours
5.00 7.49 9.90
2.50 7.46 9.89
1.25 7.45 9.84
0.625 7.46 9.90
0.313 7.49 9.88
Control 7.46 9.96

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Please refer to information on materials and methods

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks (sterile)
- Type (delete if not applicable): sealed with cotton wool plugs
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Agitation: Test containers were placed on a rotary shaker and oscillated at appr. 100 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: 10010 cells/mL
- Control end cells density: Mean 5142808 cells/mL
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 8
- No. of vessels per vehicle control (replicates): None

GROWTH MEDIUM
- Standard medium used: Yes


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Threefold concentrated AAP medium according to the guidelines

Composition of Dilution water (OECD)

Component Concentration [mg/L]
NaNO3 76.5
MgCl2  6 H2O 36.492
CaCl2  2 H2O 13.23
MgSO4  7 H2O 44.1
K2HPO4 3.132
NaHCO3 45
H3BO3 0.5565
MnCl2  4 H2O 1.246
ZnCl2 9.81 x 10-3
CoCl2  6 H2O 4.29 x 10-3
CuCl2  2 H2O 0.036 x 10-3
NaMoO4  2 H2O 21.78 x 10-3
FeCl3  6 H2O 0.4794
Na2-EDTA 0.9
pH 7.5  0.1

The pH of the test medium has to be 7.5  0.1 and was adjusted prior to testing with addition of 1 M NaOH and HCl.


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality:35 - 45 µE x m-2 x s-1 (nominal), mean: 42.4 µE x m-2 x s-1, measured values: 37.2 - 44.9 µE x m-2 x s-1 (measured, n = 32, CV = 5.19%)



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The cell density was measured daily via Chlorophyll a fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal. No self-fluorescence was found in the definitive test at the nominal loading level of 100 % of the saturated solution.


TEST CONCENTRATIONS
- Range finding study: None

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

>= 5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

>= 5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

>= 5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

> 5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Biomass, Growth Rate, Inhibition of Yield

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

> 5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Biomass, Growth Rate, Inhibition of Yield

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

> 5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Biomass, Growth Rate, Inhibition of Yield

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Biomass, Growth Rate, Inhibition of Yield

Duration:

96 h

Dose descriptor:

NOELR

Effect conc.:

>= 5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

96 h

Dose descriptor:

NOELR

Effect conc.:

>= 5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

96 h

Dose descriptor:

NOELR

Effect conc.:

>= 5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

96 h

Dose descriptor:

LOELR

Effect conc.:

> 5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Biomass, Growth Rate, Inhibition of Yield

Duration:

96 h

Dose descriptor:

EL10

Effect conc.:

> 5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Biomass, Growth Rate, Inhibition of Yield

Duration:

96 h

Dose descriptor:

EL20

Effect conc.:

> 5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Biomass, Growth Rate, Inhibition of Yield

Duration:

96 h

Dose descriptor:

EL50

Effect conc.:

> 5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Biomass, Growth Rate, Inhibtion of Yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: Microscopic evaluation of the cells at the start of the incubation period, after 24, 48, 72 hours and at test end revealed no morphological abnormalities in any test item loading rate or control.
- 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:
Current Study Valid Range (average ± 3 x SD)
Biomass inhibition
EbC50 0.284 0.442 ± 0.278
95 %
confidence interval 0.236 – 0.327
Growth Rate inhibition
ErC50 0.749 0.821 ± 0.388
95 %
confidence interval 0.715 - 0.788
Yield inhibition
EyC50 0.282 0.429 ± 0.261
95 %
confidence interval 0.239 - 0.318

Reported statistics and error estimates:

EC-values and statistical analysis:

As no effects of the test item on biomass, growth rate and yield were observed, EL10-, EL20- and EL50-values were deduced directly from the results.



NOEC/LOEC values:
The NOEL / LOEL was determined by calculation of statistical
significance of biomass integrals, growth rate and yield. As a standard, One Way Analysis of Variance (ANOVA) was used for NOEL/LOEL 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 tests are 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) is  = 0.05.

Cell Densities (Nominal test item loading rates)

Nominal test item loading rate	Replicate	Cell density [cells/mL]
[mg/L]	No.	0 hours	24 hours	48 hours	72 hours	96 hours
5.00	1	10010	57013	401835	2043029	6039986
	2	10010	54805	377524	2016247	5686371
	3	10010	66860	440106	2213753	6375538
	4	10010	63545	368254	2085679	5951677
	Mean	10010	60556	396930	2089677	6013393
2.50	1	10010	60171	394810	2042903	5082879
	2	10010	53080	366887	1973786	5129543
	3	10010	59666	351797	1986706	5105584
	4	10010	66377	402939	2017941	5678468
	Mean	10010	59824	379108	2005334	5249119
1.25	1	10010	48414	355748	1569556	3900105
	2	10010	51136	379318	2014993	5181350
	3	10010	67982	454394	2285504	5957823
	4	10010	75101	480072	1988713	5261882
	Mean	10010	60658	417383	1964692	5075290
0.625	1	10010	70457	453968	2293721	4811427
	2	10010	61767	440796	2153667	5155509
	3	10010	73797	498963	2165521	5752352
	4	10010	71379	490032	2425433	5549140
	Mean	10010	69350	470940	2259586	5317107
0.313	1	10010	60409	440031	1846653	5133682
	2	10010	58941	395349	1935840	5325982
	3	10010	60666	462134	2468835	6154137
	4	10010	80504	496429	2392505	6560751
	Mean	10010	65130	448486	2160958	5793638
Control	1	10010	47125	447394	1586804	4618500
	2	10010	49994	386192	1708355	4590151
	3	10010	62403	488677	2029732	5471492
	4	10010	62378	460378	1988713	5005357
	5	10010	60704	597521	2054632	4908643
	6	10010	80934	627903	2716265	6182110
	7	10010	69742	492979	2306767	4908016
	8	10010	73499	485265	2415586	5458196
	Mean	10010	63347	498289	2100857	5142808

Section-by-Section and Average Specific Growth Rates of the Control Group
(0 – 96 hours)

	Replicate No.	Specific growth rate [d^-1]				Mean (0 - 96 hours)	SD ±	CV [%]	Mean CV [%]
		section-by-section
		0 - 24 hours	24 - 48 hours	48 - 72 hours	72 - 96 hours
Control	1	1.55	2.25	1.27	1.07	1.53	0.517	33.7	33.8
	2	1.61	2.04	1.49	0.988	1.53	0.434	28.4
	3	1.83	2.06	1.42	0.992	1.58	0.470	29.8
	4	1.83	2.00	1.46	0.923	1.55	0.476	30.6
	5	1.80	2.29	1.24	0.871	1.55	0.624	40.3
	6	2.09	2.05	1.47	0.822	1.61	0.596	37.1
	7	1.94	1.96	1.54	0.755	1.55	0.563	36.3
	8	1.99	1.89	1.61	0.815	1.58	0.533	33.8
					Mean	1.56
					SD ±	0.03
					CV [%]	1.61

Microscopic evaluation of the cells at the start of the incubation period, after 24, 48, 72 hours and at test end revealed no morphological abnormalities in any test item loading rate or control. The environmental conditions (pH-value, water temperature, light intensity) were within the acceptable limits

Validity criteria fulfilled:

yes

Conclusions:

In this study Disperse Yellow DYLA 1306 was found to not inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata with the following effect values (nominal test item loading rates): The EL50-values for inhibition of biomass (EbL50), growth rate (ErL50) and yield (EyL50) after 72 hours were > 5.00 mg/L. The EL50-values for inhibition of biomass (EbL50), growth rate (ErL50) and yield (EyL50) after 96 hours were > 5.00 mg/L. The NOEL-values for inhibition of biomass, growth rate and yield after 72 and 96 hours were 5.00 mg/L.

Executive summary:

The toxicity of Disperse Yellow DYLA 1306 (batch number:EAG-146-1.0.0) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201, Commission Regulation (EC) No. 761/2009,C.3 and EPA Guideline 712-C-006: OCSPP 850.4500 at Dr.U.Noack-Laboratorien in 31157 Sarstedt, Germany from 2015-03-08 to 2015-03-13 with the definitive exposure phase fom 2015-03-09 to 2015-03-13.The aim of the study was to assess the effects on growth rate and yield over a period of 96 hours.

The study was conducted under static conditions with an initial cell density of 10010 cells/mL. The saturated solution and further four dilution levels were tested in a geometrical series with a dilution factor of 2:6.25 - 12.5 - 25.0 - 50.0-100 % of the saturated solution, corresponding to the nominal loading rates 0.313 - 0.625 - 1.25 - 2.50 - 5.00 mg/L. Four replicates were tested for each test item loading rate and eight replicates for the control. The environmental conditionswere within the acceptable limits.

No recovery phase was performed, because no loading level was inhibited by more than 50%.

The concentrations of the test item Disperse Yellow DYLA1306 were analytically verified via HPLC-UV at the start of the saturated solution and the control. The recovery rates ofthe test item at the start were below the limit of quantification.

All effect values were based on the nominal loading rates of the test item Disperse Yellow DYLA1306. The results are summarized in the following table:

 

NOEL, LOEL, EL-Values and 95 % Confidence Intervals of Disperse Yellow DYLA 1306

    based on nominal test item loading rates

 

	Inhibition of Biomass Integrals Nominal test item loading rates [mg/L]
	0 - 72 hours	0 - 96 hours
NOEL	5.00	5.00
LOEL	> 5.00	> 5.00
EbL10	> 5.00	> 5.00
EbL20	> 5.00	> 5.00
EbL50	> 5.00	> 5.00
	Inhibition of Growth Rate Nominal test item loading rates [mg/L]
NOEL	5.00	5.00
LOEL	> 5.00	> 5.00
ErL10	> 5.00	> 5.00
ErL20	> 5.00	> 5.00
ErL50	> 5.00	> 5.00
	Inhibition of Yield Nominal test item loading rates [mg/L]
NOEL	5.00	5.00
LOEL	> 5.00	> 5.00
EyL10	> 5.00	> 5.00
EyL20	> 5.00	> 5.00
EyL50	> 5.00	> 5.00

Description of key information

No adverse effects at the limit of solubility

Additional information

The toxicity of Disperse Yellow DYLA 1306 to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201, Commission Regulation (EC) No. 761/2009,C.3 and EPA Guideline 712-C-006: OCSPP 850.4500.

The aim of the study was to assess the effects on growth rate and yield over a period of 96 hours.

The study was conducted under static conditions with an initial cell density of 10010 cells/mL. The saturated solution and further four dilution levels were tested in a geometrical series with a dilution factor of 2:6.25 - 12.5 - 25.0 - 50.0-100 % of the saturated solution, corresponding to the nominal loading rates 0.313 - 0.625 - 1.25 - 2.50 - 5.00 mg/L. Four replicates were tested for each test item loading rate and eight replicates for the control. The environmental conditionswere within the acceptable limits.

No recovery phase was performed, because no loading level was inhibited by more than 50%.

The concentrations of the test item Disperse Yellow DYLA1306 were analytically verified via HPLC-UV at the start of the saturated solution and the control. The recovery rates ofthe test item at the start were below the limit of quantification.

All effect values were based on the nominal loading rates of the test item Disperse Yellow DYLA 1306.The results are summarized in the following table:

 

NOEL, LOEL, EL-Values and 95 % Confidence Intervals of Disperse Yellow DYLA 1306

    based on nominal test item loading rates

 

	Inhibition of Biomass Integrals Nominal test item loading rates [mg/L]
	0 - 72 hours	0 - 96 hours
NOEL	5.00	5.00
LOEL	> 5.00	> 5.00
EbL10	> 5.00	> 5.00
EbL20	> 5.00	> 5.00
EbL50	> 5.00	> 5.00
	Inhibition ofGrowth Rate Nominaltest item loading rates [mg/L]
NOEL	5.00	5.00
LOEL	> 5.00	> 5.00
ErL10	> 5.00	> 5.00
ErL20	> 5.00	> 5.00
ErL50	> 5.00	> 5.00
	Inhibition ofYield Nominal test item loading rates [mg/L]
NOEL	5.00	5.00
LOEL	> 5.00	> 5.00
EyL10	> 5.00	> 5.00
EyL20	> 5.00	> 5.00
EyL50	> 5.00	> 5.00