2,2'-[[5-acetamido-4-[(2-chloro-4,6-dinitrophenyl)azo]-2-methoxyphenyl]imino]diethyl diacetate

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:

2015-03-15 to 2015-03-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Reference substance name:

methyl 2-({4-[2-(2-bromo-4,6-dinitrophenyl)diazen-1-yl]-5-acetamido-2-methoxyphenyl}(2-methoxy-2-oxoethyl)amino)acetate

EC Number:

808-279-8

Cas Number:

88938-51-6

Molecular formula:

C21H21BrN6O10

IUPAC Name:

methyl 2-({4-[2-(2-bromo-4,6-dinitrophenyl)diazen-1-yl]-5-acetamido-2-methoxyphenyl}(2-methoxy-2-oxoethyl)amino)acetate

Test material form:

solid: particulate/powder

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

-Concentrations: The saturated solution and further four dilution levels 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 concentrations 0.313 - 0.625 - 1.25 - 2.50 - 5.00 mg/L. The concentration levels were based on the sponsor’s proposal.

- Sampling method: Analytical evaluation of the saturated solution of Structural Analogue 01 and the control was carried out via
HPLC-UV from freshly prepared media after 0 hours. Separate replicates for the test item analysis at the beginning of exposure were prepared without algae. The recovery rates of the test item at the start were below the limit of quantification. Therefore, no analytical evaluation after 96 h was carried out.


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) with a magnetic stirrer. Undissolved particles were removed by membrane filtration (membrane filter 0.2 µm, RC, MACHEREY-NAGEL).
Application Application was carried out by adding appropriate volumes of the stock solution to the replicate test vessels.
- Differential loading: The saturated solution and further four dilution levels of the
(nominal) 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 concentrations 0.313 - 0.625 - 1.25 - 2.50 - 5.00 mg/L. The concentration levels were based on the sponsor’s proposal.
- Dilution water: Threefold concentrated AAP medium according to the guidelines, composition of dilution water see below -Controls: Eight replicates (without test item) were tested under the same test conditions as the test replicates.

Test organisms

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, standard growth medium
- Any deformed or abnormal cells observed: No

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

None

Test conditions

Hardness:

Not measured

Test temperature:

Mean: 24.8 °C, Min: 24.5 °C, Max:25.6 °C

pH:

Nomina
test item concentration pH-value
[mg/L] Start; 0 h End; 96 h
5.00 7.94 9.86
2.50 7.86 9.87
1.25 7.71 9.84
0.625 7.62 9.95
0.313 7.58 9.90
Control 7.55 9.67

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 6289107 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: According to the guideline (OECD)

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
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), 40.7 µE x m-2 x s-1 (measured, n = 32, CV = 6.69%)



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

- Microscopic evaluation : Microscopic evaluation of the cells was carried out daily. The cells were checked for any unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation and adherence of algae to test containers or aggregation of algae cells.


TEST CONCENTRATIONS
- Range finding study: No

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

growth rate, yield

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

growth rate, yield

Effect concentrations 3

Duration:

96 h

Dose descriptor:

EC10

Effect conc.:

> 5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

growth rate, yield

Effect concentrations 4

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

> 5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

growth rate, 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 concentration 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:
Biomass inhibition 0.284 (CI 0.236 - 0.327)
Rate-related inhibition: 0.749 (CI 0.715 - 0.788) mg/L
Yield Inhibition: 0.282 (CI 0.239 - 0.318) mg/L

Reported statistics and error estimates:

EC-values and statistical analysis:

EC-values and statistical analyses: As no effects of the test item on biomass, growth rate and yield were observed, EC10-, EC20- and EC50-values were deduced directly from the results.

NOEC, LOEC and statistical analyses:The NOEC / LOEC was determined by calculation of statistically significant differences of biomass integrals, growth rate and yield between test substance treated and control groups. As a 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 tests are 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) is  = 0.05.
t.

Any other information on results incl. tables

          

  Cell Densities (nominal test item concentration)

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	58411	498743	2591246	6866931
	2	10010	59681	561087	3191225	7165227
	3	10010	66458	539104	3354673	7782016
	4	10010	65295	573343	2990709	6377464
	Mean	10010	62461	543069	3031963	7047910
2.50	1	10010	50229	523141	2356736	6188928
	2	10010	49204	603310	3236070	6996761
	3	10010	62730	572025	2763537	7334571
	4	10010	67565	589374	4046977	8614812
	Mean	10010	57432	571963	3100830	7283768
1.25	1	10010	46783	472652	2336163	5794042
	2	10010	44553	469641	2439526	6148536
	3	10010	62068	586790	3186521	6651550
	4	10010	59481	587103	3000117	7190942
	Mean	10010	53221	529047	2740582	6446268
0.625	1	10010	53880	503360	2584346	7042798
	2	10010	43434	491179	2337355	6235466
	3	10010	66185	569793	2803678	7103887
	4	10010	57311	599409	2672593	6870819
	Mean	10010	55203	540935	2599493	6813243
0.313	1	10010	57279	504978	2507389	6268331
	2	10010	55329	501603	2352282	6460631
	3	10010	44923	435057	2283918	4154792
	4	10010	59531	483063	2516170	6373575
	Mean	10010	54266	481175	2414940	5814332
Control	1	10010	52459	485509	2459596	6362160
	2	10010	40110	373943	1798653	5739601
	3	10010	56442	473919	2251868	6176634
	4	10010	42841	452707	2285925	6159825
	5	10010	63457	580907	2638160	6331929
	6	10010	49972	474834	3377002	6872074
	7	10010	45093	454902	2355356	5934410
	8	10010	41596	458502	2746791	6736222
	Mean	10010	48996	469403	2489169	6289107

 

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	1.66	2.23	1.62	0.950	1.61	0.521	32.3	34.5
	2	1.39	2.23	1.57	1.16	1.59	0.461	29.1
	3	1.73	2.13	1.56	1.01	1.61	0.464	28.9
	4	1.45	2.36	1.62	0.991	1.61	0.568	35.3
	5	1.85	2.21	1.51	0.876	1.61	0.569	35.3
	6	1.61	2.25	1.96	0.710	1.63	0.669	41.0
	7	1.51	2.31	1.64	0.924	1.60	0.570	35.7
	8	1.42	2.40	1.79	0.897	1.63	0.632	38.8
					Mean	1.61
					SD ±	0.02
					CV [%]	0.93

SD = Standard deviation CV = Coefficient of variation

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	1.66	2.23	1.62	1.83	0.339	18.5	21.5
	2	1.39	2.23	1.57	1.73	0.444	25.7
	3	1.73	2.13	1.56	1.81	0.292	16.2
	4	1.45	2.36	1.62	1.81	0.481	26.6
	5	1.85	2.21	1.51	1.86	0.351	18.9
	6	1.61	2.25	1.96	1.94	0.322	16.6
	7	1.51	2.31	1.64	1.82	0.431	23.7
	8	1.42	2.40	1.79	1.87	0.493	26.3
				Mean	1.83
				SD ±	0.06
				CV [%]	3.30

SD = Standard deviation CV = Coefficient of variation

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study, Structural Analogue 01 was found not to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata with the following effect values (nominal test item loading rates): The EL10 and 50-values for inhibition of biomass, growth rate and yield after 72 and 96 hours were > 5.00 mg/L.

Executive summary:

The toxicity of Structural Analogue 01 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 (2009) and EPA Guideline 712-C-006: OCSPP 850.4500 (2012) at Dr.U.Noack-Laboratorienin 31157 Sarstedt, Germanyfrom 2015-03-15 to 2015-03-20 with the definitive exposure phase from 2015-03-16 to 2015-03-20.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 solution¹and 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 itemloading rateand eight replicates for the control. The environmental conditionswere within the acceptable limits.

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

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

All effect values were based on the nominal loading ratesof the test item.

EL-Values of the test item

based on nominal test item loading rates

 

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