5-({4-chloro-6-[ethyl(phenyl)amino]-1,3,5-triazin-2-yl}amino)-4-hydroxy-3-[(2-sulfophenyl)diazenyl]naphthalene-2,7-disulfonic acid, lithium 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 July 24, 2017 to October 11, 2017

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):

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

Details on test organisms:

- Source (laboratory, culture collection): Collection of Algal Cultures, Inst. Plant Physiology, and University of Gӧttingen, GERMANY.

Water media type:

freshwater

Total exposure duration:

72 h

pH:

7.54-8.97

Nominal and measured concentrations:

Nominal Concentrations: 1.0, 3.1, 9.8, 31.3 and 100.0 mg/L

Details on test conditions:

- Test vessel: 250 mL Erlenmeyer flask
- Test volume: 100 mL
- Type of cover: covered with air-permeable stoppers
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Light intensity and quality: about 8335 lux

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Remarks:

ErC50

Effect conc.:

> 100 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.:

14.55 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

EC50

Remarks:

EbC50

Effect conc.:

27.67 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Results with reference substance (positive control):

The 72-h ErC50 was 0.88 mg/L.
The 72-h EyC50 was 0.54 mg/L.
The 72-h EbC50 was 0.64 mg/L.

Table 1. Results of the Preliminary Range-Finding Test

Nominal concentrations (mg/L)	Untreated control	0.1	1	10	100
Average of cell number at 72 hours (× 10^4cell/mL)	68.33	68.5	67.5	34.50	16.5

*The test was started (0 hours) by inoculation of biomass of approximately 10⁴algal cells per mL test medium.

 

For the definitive test, the cell density in the control cultures increased by the factor of 68.67 within three days. The mean coefficient of variation for section-by-section specific growth rates (Day 0-1, Day 1-2 and Day 2-3) in the control cultures was 15.91 %. The coefficient of variation of average specific growth rates during the whole test period (day 0-3) in the control cultures was 1.62%.

 

Table 2. Growth Rates (μ) and Percentage Inhibition of μ during the Test Period

Nominal Concentration (mg/L)	Growth rates (μ) and % inhibitionof μ
	0-24 h		0-48 h		0-72 h
	μ	%	μ	%	μ	%
Control	0.0533^+	0.0	0.0601	0.0	0.0587^+	0.0
1.0	0.0538	-0.8	0.0606^+	-0.7	0.0586	0.2
3.1	0.0498	6.7	0.0564	6.2	0.0539*	8.2
9.8	0.0458	14.1	0.0528*	12.2	0.0487*	17.1
31.3	0.0401^+	24.7	0.0506*	15.9	0.0447*^+	23.9
100.0	0.0458	14.1	0.0517*	14.0	0.0393*	33.0

*: statistically significantly different compared to the control values (Bonferroni t-Test; α = 0.05)

+: at these values the rounding of the EXCEL and TOXSTAT software was different. The table contains the values calculated with EXCEL.

 

Table 3. Area under the Growth Curves (A) and Percentage Inhibition of A during the Test Period

Nominal Concentration (mg/L)	Area under the Growth Curves (A) and Percentage Inhibition of A
	0-24 h		0-48 h		0-72 h
	A	%	A	%	A	%
Control	32.0	0.0	268.0	0.0	1284.0	0.0
1.0	32.0	0.0	272.0	-1.5	1284.0	0.0
3.1	28.0	12.5	224.0	16.4	964.0*	24.9
9.8	24.0	25.0	188.0*	29.9	716.0*	44.2
31.3	20.0	37.5	164.0*	38.8	576.0*	55.1
100.0	24.0	25.0	180.0*	32.8	504.0*	60.7

*: statistically significantly different compared to the control values (Bonferroni t-Test; α = 0.05)

 

Table 4. Yield (Y) and Percentage Inhibition of Y during the Test Period

Nominal Concentration (mg/L)	Yield (Y) and % inhibition of Y
	0-72 h
	Y	%
Control	67.7	0.0
1.0	67.0	1.0
3.1	47.7*	29.6
9.8	32.3*	52.2
31.3	24.0*	64.5
100.0	16.0*	76.4

*: statistically significantly different compared to the control values (Bonferroni t-Test; α = 0.05)

 

Table 5. Influence of CJ305 on the Growth of Pseudokirchneriella subcapitata

Parameter (0-72h)	Growth rate (r) (mg/L)	Yield (y) (mg/L)	Biomass (b) (mg/L)
	Calculation based on the nominal concentrations
EC50	>100	14.55	27.67
95 % conf. limits	Not determined	11.26-18.79	19.89-38.49
NOEC	3.1	1.0	1.0
LOEC	9.8	3.1	3.1

Validity criteria fulfilled:

yes

Conclusions:

According to OECD 201 test method, the ErC50, the EyC50 and the EbC50 of CJ305 was >100.00 mg/L, 14.55 mg/L and 27.67 mg/L respectively.

Executive summary:

This test using the procedures outlined in the CiToxLAB Study Plan for 16/377-022AL and OECD 201 (OECD, 2011). The results of this OECD 201 test for CJ305 show that test validity criteria was met. In the alga growth inhibition test (definitive test), the test article concentrations applied were 1.0, 3.1, 9.8, 31.3 and 100.00 mg/L. Under the test conditions, the E_rC₅₀, the E_yC₅₀and the E_bC₅₀of CJ305 was >100.00 mg/L, 14.55 mg/L and 27.67 mg/L respectively.

Description of key information

Respectively,the E_rC₅₀, the E_yC₅₀and the E_bC₅₀of CJ305 was >100.00 mg/L, 14.55 mg/L and 27.67 mg/LforPseudokirchneriella subcapitata(OECD TG201).

 

Additional information

The cell concentration in the untreated control was increased exponentially by a factor of at least 16 within the 72-hour test period. The mean coefficient of variation for specific growth rates (Day 0-1, Day 1-2 and Day 2-3) in the control cultures 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. Based on the preliminary range-finding test, the nominal concentration were 1.0, 3.1, 9.8, 31.3 and 100.0 mg/L in the definitive test. As the measured concentrations deviated not more than 20 percent from the nominal, biological results are based on the nominal concentrations. There were no any observed morphological deviations during the experiment.Under the test conditions, the E_rC₅₀, the E_yC₅₀ and the E_bC₅₀ of CJ305 was >100.00 mg/L, 14.55 mg/L and 27.67 mg/L respectively.