2,2'-[[3-chloro-4-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]phenyl]imino]bisethyl bis(hydrogen sulphate), potassium sodium salt

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:

2017-07-17 to 2018-02-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

28 July 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-[[3-chloro-4-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]phenyl]imino]bisethyl bis(hydrogen sulphate), potassium sodium salt

EC Number:

287-719-5

EC Name:

2,2'-[[3-chloro-4-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]phenyl]imino]bisethyl bis(hydrogen sulphate), potassium sodium salt

Cas Number:

85567-07-3

Molecular formula:

C36H42Cl2KN6NaO28S8

IUPAC Name:

2,2'-[[3-chloro-4-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]phenyl]imino]bisethyl bis(hydrogen sulphate), potassium sodium salt

Test material form:

solid

Details on test material:

Batch: 1283067001

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 100, 16.7, 2.78, 0.463, 0.0772, 0.0129 mg/L
- Sampling method: taken in the main test at 0 hours (initial value) from fresh test solutions and after 24 hours, 48 hours and 72 hours from aged solutions from all test item concentrations and control. For each sampling also a retain sample was taken.
- Sample storage conditions before analysis: mixed with formic acid (0.5% v/v final concentration) immediately after sampling and stored frozen until analysis

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Stock solution: weight necessary amount of test item in volumetric flask and added test medium up to the benchmark, then homogenised by shaking and one minute of sonication. Lower test solutions: dilution appropriate solution with test medium
- Test concentration: 00, 16.7, 2.78, 0.463, 0.0772, 0.0129 mg/L and control

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: SAG 61.81
- Source (laboratory, culture collection): MBM Sciencebridge GmbH, Hans-Adolf-Krebs-Weg 1, D-37077 Göttingen, Germany.
- Method of cultivation: grown semi-continuously in sterile cultures in the laboratory. Old medium is periodically replaced by fresh mineral solution.
- Culture conditions: Illumination: continuously (approx. 4440 - 8880 lux at cell culture level or 60 - 120 µEm-2s-1); Temperature: 21 - 24 °C; Culture flasks: 100 mL Erlenmeyer flasks; CO2 supply by shaking on a rotating shaker, approximately 105 rpm

ACCLIMATION
- Acclimation period: 3 to 4 day
- Culturing media and conditions (same as test or not): same as test

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

22.7 – 23.0 °C

pH:

7.29 to 7.47 at 0 h and 8.03 to 8.33 at 72 h

Nominal and measured concentrations:

Nominal concentrations: 100, 16.7, 2.78, 0.463, 0.0772, 0.0129 mg/L
Measured initial concentration: 92.69, 14.75, 2.154 mg/L, < LOQ, < LOD, not determined

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks (100 mL) with aluminium caps
- Type : closed, aluminium caps
- Material, size, headspace, fill volume: volume: 100 mL, fill volume: ~ 50 mL
- Initial cells density: 0.5E+04 cells/mL
- CO2 supplied: by continuous agitation
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, As recommended in OECD TG 201

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: Continuously from the side
- Light intensity and quality: 88.8 µEm-2s-1 (mean)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: fluorescence measurement at 24, 48 and 72 hours
- Other: morphological appearance: at the end of the test; pH-value: at t = 0 h and t = 72 h; temperature: measured continuously and recorded at hours 0, 24, 48 and 72 h; light intensity: measured once a year, and confirmed for one representative position at test start

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 6
Range finding study
- Test concentrations: 100, 10.0, 1.00, 0.100, 0.0100 mg/L and control
- Results used to determine the conditions for the definitive study: After 72 h at termination of the test a flat dose response relation was observed for the inhibition of growth rate and yield from nominal concentrations of 0.0100 to 100 mg/L. The inhibition of growth rate peaked in 74.3 % and the inhibition of yield peaked in 95.5 % at a nominal concentration of 100 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

44.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.36 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.077 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Morphology evaluation: normal for the control and up to and including a nominal test item concentration of 16.7 mg/L. No cells were observed at 100 mg/L nominal test item concentration.
- Physicochemical Data: pH and temperature during the test, light intensity were within the range specified by OECD 201.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50: growth rate 1.23 mg/L, yield 0.685 mg/L, biomass intergral 0.639 mg/L

Reported statistics and error estimates:

EC50: Probit analysis following Gompertz distribution for growth rate and following normal distribution for yield
NOEC following Dunnetts-t-test for growth rate and following Bonferroni-Holms corrected Welch test for yield (left-sided, p<0.05)

Any other information on results incl. tables

Percentage inhibition of growth rate and yield observed in the non-GLP range-finding test:

Conc.  [mg/L]	% Inhibition at test end (72 h)
	Growth rate	Yield
Control	0	0
0.0100	-7.5	-34.8
0.100	4.7	16.6
1.00	12.0	37.4
10.0	26.5	64.4
100	74.3	95.5

Biological results, main test:

Nominal concentration (mg/L)	Average cell number (10^4/mL)
	0h	24h	48h	72h
Control	0.54	1.83	8.13	40.24
0.0129	0.54	1.72	6.91	35.37
0.0772	0.54	1.88	8.41	41.15
0.463	0.54	1.53	5.99	27.91
2.78	0.54	1.41	5.23	19.32
16.7	0.54	1.35	3.55	11.2
100	0.54	0.66	1.12	2.27

Percentage inhibition of growth rate:

Conc.	% Inhibition of growth rate
[mg/L]	0 h – 24 h	0 h – 48 h	0 h – 72 h
Control	0.0	0.0	0.0
0.0129	4.6	5.7	2.8^1)
0.0772	-3.3	-1.4	-0.5^2)
0.463	13.9	10.8	8.3 *
2.78	21.1	15.8	17.0 *
16.7	23.8	30.2	29.6 *
100	84.4	73.4	66.6 *

¹⁾Value was omitted for EC_{10, 20, 50}-calculation

²⁾Value was set to zero for EC_{10, 20, 50}-calculation

* Statistically significant different to the control

Percentage inhibition of yield:

Conc.	% Inhibition of yield
[mg/L]	0 h – 24 h	0 h – 48 h	0 h – 72 h
Control	0.0	0.0	0.0
0.0129	8.5	16.0	12.3^1)
0.0772	-3.9	-3.8	-2.3^2)
0.463	23.3	28.1	31.1 *
2.78	32.6	38.1	52.7 *
16.7	37.2	60.3	73.1 *
100	90.7	92.3	95.6 *

¹⁾Value was omitted for EC_{10, 20, 50}-calculation

²⁾Value was set to zero for EC_{10, 20, 50}-calculation

* Statistically significant different to the control

Analytical results:

Test item nominal concentration [mg/L]	Sampling	Test item measured concentration [mg/L]]
			% of nominal
Control	0 h fresh	< LOD	-
	24 h aged	< LOD	-
	48 h aged	< LOD	-
	72 h aged	< LOD	-
0.0772	0 h fresh	< LOD	-
	24 h aged	< LOD	-
	48 h aged	< LOD	-
	72 h aged	< LOD	-
0.463	0 h fresh	< LOQ	-
	24 h aged	< LOQ	-
	48 h aged	< LOQ	-
	72 h aged	< LOQ	-
2.78	0 h fresh	2.277	82
	24 h aged	2.251	81
	48 h aged	2.096	75
	72 h aged	1.992	72
16.7	0 h fresh	15.142	91
	24 h aged	14.754	88
	48 h aged	14.624	88
	72 h aged	14.495	87
100	0 h fresh	91.658	92
	24 h aged	92.694	93
	48 h aged	92.694	93
	72 h aged	93.731	94

 - = not calculated; LOD = 0.195 mg/L; LOQ = 0.650 mg/L

NOEC, LOEC, ECx-values and 95 % Confidence Intervals (0 - 72 hours):

	Test item[mg/L]
	nominal
ErC10(Growth rate)	1.36
95 % confidential limits	0.613 – 2.40
ErC20	5.47
95 % confidential limits	3.24 – 8.18
ErC50	44.7
95 % confidential limits	31.8 – 65.7
EyC10(Yield)	0.156
95 % confidential limits	0.00499 – 0.591
EyC20	0.416
95 % confidential limits	0.0331 – 1.29
EyC50	2.72
95 % confidential limits	0.778 – 9.17
NOEC	0.0772
LOEC	0.463

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Significant inhibitory effects were determined for growth rate and for yield at test item concentrations of 0.436 mg/L (nominal) and above. Following effect levels were determined based on nominal concentrations: NOEC = 0.0772 mg/L, EC10 (growth rate) = 1.36 mg/L, EC50 (growth rate) = 44.7 mg/L.

Executive summary:

The effects of test item on the growth of the single cell green alga Pseudokirchneriella subcapitata were determined according to OECD 201.

Following a static non-GLP range-finding test with nominal test item concentrations of 100, 10.0, 1.00, 0.100, 0.0100 mg/L and control, a static main test with nominal test item concentrations of 100, 16.7, 2.78, 0.463, 0.0772, 0.0129 mg/L and control was performed. Six replicates were employed for the control and three for each test item concentration.

The test concentrations were analytically verified. The measured concentrations of test item in the samples mostly remained within 80 – 120 % of the start concentration. Therefore the effect levels were expressed based on nominal concentrations.

Significant inhibitory effects were determined for growth rate and for yield at test item concentrations of 0.436 mg/L (nominal) and above. Following effect levels were determined based on growth rate and nominal concentrations: NOEC = 0.0772 mg/L, EC10 (growth rate) = 1.36 mg/L, EC50 (growth rate) = 44.7 mg/L. When yield inhibition is considered, following effect levels were observed: EC10 (yield) = 0.156 mg/L, EC50 (yield) = 2.72 mg/L.