2-substitutedalkyl N-{3-acetamido-4-[(2-cyano-4,6-disubstitutedphenyl)diazenyl]phenyl}-N-benzyl-β-alaninate

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:

2015-03-08 to 2015-03-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Test item: Disperse Blue 1092

Sampling and analysis

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 Blue 1092 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).

Test solutions

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. Undissolved particles were removed by membrane filtration (membrane filter 0.2 µm, RC, MACHEREY-NAGEL).

- 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

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis 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

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None

Test conditions

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 7.50 10.1
2.50 7.43 10.1
1.25 7.42 10.1
0.625 7.43 10.1
0.313 7.44 9.91
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 = 31, 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 5.00 mg/L.


TEST CONCENTRATIONS
- Range finding study: None

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Biomass, Growth Rate, Inhibition of Yield

Effect concentrations 2

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

Effect concentrations 3

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

Effect concentrations 4

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

Effect concentrations 5

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

Effect concentrations 6

Duration:

96 h

Dose descriptor:

NOELR

Effect conc.:

5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Biomass, Growth Rate, Inhibition of Yield

Effect concentrations 7

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

Effect concentrations 8

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

Effect concentrations 9

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

Effect concentrations 10

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) and DUNNETT’s test were 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.

Any other information on results incl. tables

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	60729	424426	2080159	6213219
	2	10010	60130	442904	2176121	5979524
	3	10010	66499	514743	2481316	7757511
	4	10010	61287	506401	2448953	6864880
	Mean	10010	62161	472119	2296637	6703784
2.50	1	10010	48661	371165	1905609	5507619
	2	10010	51199	350806	1850604	5482656
	3	10010	54429	379117	1990219	6246962
	4	10010	54940	423799	2201146	6806550
	Mean	10010	52307	381222	1986895	6010947
1.25	1	10010	56332	414090	1801745	5088273
	2	10010	45303	296728	1620422	5380925
	3	10010	55705	397959	1866221	5889960
	4	10010	56586	403591	2067929	6594055
	Mean	10010	53482	378092	1839079	5738303
0.625	1	10010	53488	430786	1841447	4929466
	2	10010	57420	441022	2020512	5630048
	3	10010	65775	487234	1996365	6245332
	4	10010	54501	459688	2207167	6739063
	Mean	10010	57796	454683	2016373	5885977
0.313	1	10010	52732	432668	1919282	5113737
	2	10010	50728	384373	1731499	5366374
	3	10010	64069	502425	2215446	6236175
	4	10010	57333	489015	2197383	7210592
	Mean	10010	56216	452120	2015903	5981720
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 – 72 hours)

	Replicate No.	Specific growth rate [d^-1]			Mean (0 - 72 hours)	SD ±	CV [%]	Mean CV [%]
		section-by-section
		0 - 24 hours	24 - 48 hours	48 - 72 hours
Control	1	1.55	2.25	1.27	1.69	0.507	30.0	19.1
	2	1.61	2.04	1.49	1.71	0.293	17.1
	3	1.83	2.06	1.42	1.77	0.321	18.1
	4	1.83	2.00	1.46	1.76	0.274	15.5
	5	1.80	2.29	1.24	1.78	0.526	29.7
	6	2.09	2.05	1.47	1.87	0.350	18.7
	7	1.94	1.96	1.54	1.81	0.234	12.9
	8	1.99	1.89	1.61	1.83	0.201	11.0
				Mean	1.78
				SD ±	0.06
				CV [%]	3.32

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

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study Disperse Blue 1092 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 with 95 % confidence intervals for inhibition of biomass (EbL50), growth rate (ErL50) and yield (EyL50) after 72 hours were > 5.00 mg/L. The EL50-values with 95 % confidence intervals 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 Blue 1092 (batch number:EAG-148-1.0.0) 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, 2012 at Dr.U.Noack-Laboratorien in 31157 Sarstedt, Germany from 2015-03-08 to 2015-03-13 with the definitive exposure phase from 2015-03-09 to 2015-03-13. The aim of the study was to assess the effects on biomass integrals, 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 levels 0.313 - 0.625 - 1.25 - 2.50 - 5.00 mg/L. Four replicates were tested for each loading level 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 Blue 1092 were analytically verified via HPLC-UV at the start of the saturated solution and the control. The recovery rate of the saturated solution at the start was below the limit of quantification.

All effect values were based on the nominal loading levels of the test item Disperse Blue 1092. The results are summarized in the following table:

 

NOEL, LOEL, EL-Values and 95 % Confidence Intervals of Disperse Blue 1092

    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