Reaction products of 4-Diazo-3,4-dihydro-7-nitro-3-oxo-1-naphthalenesulfonic acid coupled with 7-Amino-4-hydroxy-2-naphthalenesulfonic acid, subsequently chelated with Chromium(3+) and cobalt(3+), further mixed with reaction products of 2,4,6-Trichloro-1,3,5-triazine and Ethanol, 2-[(4-aminophenyl)sulfonyl]-, 1-(hydrogen sulfate), sodium salts

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:

From January 12, 2018 to March 27, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Test organisms (species):

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

Details on test organisms:

- Source: Culture Collection of Algae at Goettingen University

Test type:

static

Water media type:

freshwater

Total exposure duration:

72 h

Test temperature:

22.3 to 22.4 °C

pH:

8.1-8.8

Nominal and measured concentrations:

Nominal concentrations for the definitive test: 1, 3.2, 10, 32 and 100 mg/L

Details on test conditions:

- Test vessel: 250ml glass Erlenmeyer
- Test volume: 100 mL
- No. of vessels per concentration (replicates): 3 replicates per each concentration
- No. of vessels per control (replicates): 3 replicates
- Light: 4716 lux (average)

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Remarks:

ErC50

Effect conc.:

61.75 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Remarks:

EyC50

Effect conc.:

25.86 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: algal growth

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth

Results with reference substance (positive control):

72-hours EyC50 of the reference substance was 0.79 mg/L.

Table 1. Nominal and Effective Concentrations of Test Solutions and Their Respective Deviations.

Nominal concentration (mg/L)	Mean of effective concentration of test substance (Cts^mean) (mg/L)	|Deviation| (|Dev|) (%)
Control	< LQ	nd
1	1.1539	15.39
3.2	3.0007	6.23
10	10.1608	1.61
32	31.9459	0.17
100	97.5803	2.42

nd: not determined.

<LQ: lower than the practical limit of quantification (1.01μg/mL)

 

Table 2. Daily cell counts in control replicates.

Control replicate	Cells density (cells/mL)				Exponential increase of the biomass of the control group – 72 hours
	Day 0	Day 1	Day 2	Day 3	135
1	5000	40000	200000	650000
2	5000	40000	200000	690000
3	5000	30000	220000	680000
Average	5000	36667	206667	673333

 

Table 3. Calculation of Percentage Coefficient of Variation for section-by-section specific growth rates.

Control replicate	Section-by-section specific growth rates (day^-1)			Average (day^-1)	Standard deviation (sd) (day^-1)	Coefficient of variation (%) %CV
	Day 0-1	Day 1-2	Day 2-3
1	2.0794	1.6094	1.1787	1.6225	0.4505	27.8
2	2.0794	1.6094	1.2384	1.6424	0.4215	25.7
3	1.7918	1.9924	1.1285	1.6376	0.4522	27.6
Average section-by-section %CV						27.0

Table 4. Calculation of Percentage Coefficient of Variation for Average Specific Growth Rates in The Control over The Study Duration.

Control replicate	Average specific growth rates over the study duration (day^-1)
1	1.6225
2	1.6424
3	1.6376
Average specific growth rates over the study duration (day^-1)	1.6342
sd for average specific growth rates over the study duration (day^-1)	0.0104
%CV over study duration	0.6

Table 5. Cells Concentration, Yield of Biomass and Yield Inhibition, Growth Rate, and Growth Rate Inhibition of Cells Compares to the Control at 24, 48, and 72 Hours at Each Tested Concentration, in the Definitive Test.

Test period (hours)	Nominal concentration (mg/L)	Obs*	Cells concentration (cells/mL)			Cells concentration average (cells/mL)	Yield (y) (cells/mL)	Yield inhibition (Iy) (%)	Growth rate (μ) (day^-1)	Growth rate inhibition (Iμ) (%)
			R1	R2	R3
24	Control	NM	40000	40000	30000	36667	31667	0	1.9924	0
	1	NM	40000	40000	30000	36667	31667	0	1.9924	0
	3.2	NM	40000	40000	30000	36667	31667	0	1.9924	0
	10	NM	30000	40000	40000	36667	31667	0	1.9924	0
	32	NM	30000	20000	20000	23333	18333	42	1.5404	23
	100	NM	10000	10000	10000	10000	5000	84	0.6931	65
48	Control	NM	200000	200000	220000	206667	201667	0	1.8608	0
	1	NM	210000	170000	240000	206667	201667	0	1.8608	0
	3.2	NM	180000	200000	240000	206667	201667	0	1.8608	0
	10	NM	190000	170000	190000	183333	178333	12	1.8009	3
	32	NM	100000	90000	100000	96667	91667	55	1.4809	20
	100	NM	20000	10000	10000	13333	8333	96	0.4904	74
72	Control	NM	650000	690000	680000	673333	668333	0	1.6343	0
	1	NM	680000	630000	700000	670000	665000	0	1.6326	0
	3.2	NM	710000	640000	660000	670000	665000	0	1.6326	0
	10	NM	580000	590000	680000	616667	611667	8	1.6050	2
	32	NM	270000	250000	250000	256667	251667	62	1.3128	20
	100	NM	20000	20000	20000	20000	15000	98	0.4621	72

R1, R2 and R3 = replicates

*NM = None morphological effects observed.

Validity criteria fulfilled:

yes

Conclusions:

According to OECD 201 test method, the 72h-ErC50 and the 72-EyC50 of Everzol SR98 was 61.75 mg/L and 25.86 mg/L respectively. The highest test substance concentration which no observed significant adverse effect (NOEC) on algal growth was 10mg/L and the lowest test substance concentration at which was observed significant reducing effect on growth (LOEC) was 32 mg/L.

Executive summary:

This test using the procedures outlined in the BIOAGRI Study Plan for 16264.202.078.17 and OECD 201 (OECD, 2011). The results of this OECD 201 test for Everzol SR98 show that test validity criteria was met.Under the test conditions,the 72h-ErC50 and the 72-EyC50 of Everzol SR98 was 61.75 mg/L and 25.86 mg/Lrespectively. The NOEC was 10mg/L and the LOEC was 32 mg/L.

Description of key information

Respectively,the 72h-ErC50 and the 72-EyC50 of Everzol SR98 was 61.75 mg/L and 25.86 mg/L for Pseudokirchneriella subcapitata (OECD TG201).

Key value for chemical safety assessment

EC50 for freshwater algae:

61.75 mg/L

EC10 or NOEC for freshwater algae:

10 mg/L

Additional information

The cell densitiesin the control have increased by an average factor of at least 16 within 2 days. The mean coefficient of variation for section-by-section specific growth rates in the control group were not exceed 35%. The coefficient of variation of average specific growth rates during the whole test period in replicate control groups were not exceed 7%. This test validity criteria was met. Under the test conditions,the 72h-ErC50 and the 72-EyC50 of Everzol SR98 was 61.75 mg/L and 25.86 mg/L respectively.