Octasodium 2-(6-(4-chloro-6-(3-(N-methyl-N-(4-chloro-6-(3,5-disulfonato-2-naphthylazo)-1-hydroxy-6-naphthylamino)-1,3,5-triazin-2-yl)aminomethyl)phenylamino)-1,3,5-triazin-2-ylamino)-3,5-disulfonato-1-hydroxy-2-naphthylazo)naphthalene-1,5-disulfonate

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 October 4, 1993 to October 7, 1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Purity: 66.9% w/w
Batch number: NBY 35071/15
Physical state: solid
Color: orange/red

Analytical monitoring:

yes

Details on sampling:

Nominal concentrations at 1, 2, 4, 8, 16, 32, 64, and 128 mg/L at 0 and 48 h

Vehicle:

no

Details on test solutions:

A total of 1600 mL of the highest nominal exposure concentration (128mg/L) was prepared by the direct addition of test substance to sterile culture medium.

All lower test concentrations were prepared using sterile medium, to a final volume of 1000 mL, by the addition of aliquots of the nominal 128 mg/L solution. The control consisted of culture medium only.

The nominal exposure concentrations used in this study were 1.0, 2.0, 4.0, 8.0, 16, 32, 64, and 128 mg/L together with a control.

The test solutions were all clear and exhibited an orange colouration which increased with increasing nominal concentration. Using aseptic techniques 100 m1 volumes of the appropriate test solution were dispensed to each test and blank vessel. The remaining test solutions were used for physical and chemical analysis.

Test organisms (species):

Selenastrum sp.

Details on test organisms:

The test organism was the unicellular green algae Selenastrum capricornururn Printz, (Strain ATCC 22662) from laboratory cultures maintained under axenic conditions. A culture of the algae in the exponential growth phase was used as inoculum for the test. The culture was grown in the medium, and under the environmental conditions, described for the test.

Test type:

static

Water media type:

freshwater

Total exposure duration:

72 h

Test temperature:

24.1 to 24.4°C

pH:

At the start of the test the pH ranged from 7.3 to 7,4 and at the end of the rest the range was 7.4 to 8.6

Nominal and measured concentrations:

Nominal concentrations mg/L Measured at 0 h mg/L Measured at 72 h mg/L
1 0.85 0.87
2 2 2
4 4.1 3.7
8 8.1 7.9
16 17 17
32 32 32
64 63 73
128 130 130

Details on test conditions:

TEST SYSTEM
- Test vessel: Borosilicate glass conical flask
- Type (delete if not applicable): closed with polyurethene foam bungs
- Material, size, headspace, fill volume: 250 mL nominal capacity

-Each replicate test vessel was inoculated with 0.925 mL of the inoculum culture to give a nominal cell density of 1.00 x 104 cells/mL. Three 100 mL volumes' of Coulter electrolyte, inoculated in the same manner, had a mean measured cell density of 1.03 x 104 cells/mL. This value was used for growth calculations.

- Six replicate cultures of the control, and triplicate cultures at each concentration of the test substance were employed. One blank vessel (without algal inoculum) for each control and. treatment was incubated concurrently. The position of the vessels in the incubator were re-randomized, daily, by rows.

Reference substance (positive control):

not specified

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

34 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 128 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

1. Analytical results:

The analytical results for the levels of test substance are reported in Table 1. The measured concentrations at the start of the test ranged from 85 to 106% of the nominal values. The measured concentrations in the blanks after 72 hours ranged from 87 to 114% of the nominal values (based on test substance as received). On the basis of this analysis nominal test concentrations were used for the calculation of the biological results.

Nominal concentration of test susbtance (mg/L)	Exposure time	Concentration determined	percentage of nominal
control	0.00	< 0.43	-
	72.00	< 0.43	-
1.00	0.00	0.85	85.00
	72.00	0.87	87.00
2.00	0.00	2.00	100.00
	72.00	2.00	100.00
4.00	0.00	4.10	103.00
	72.00	3.70	93.00
8.00	0.00	8.10	101.00
	72.00	7.90	99.00
16.00	0.00	17.00	106.00
	72.00	17.00	106.00
32.00	0.00	32.00	100.00
	72.00	32.00	100.00
64.00	0.00	63.00	98.00
	72.00	73.00	114.00
128.00	0.00	130.00	102.00
	72.00	130.00	102.00

2. Biological data: The algal cell densities measured at each time period and meancell densities are also shown in below table 2.

The area under the growth curve (Days 0 to 3) was calculated for each replicate culture, according to the formula given in OECD Guideline 201. These areas were examined by one-way analysis of variance, and Dunnett's procedure was used to identify significant differences (P>0.05) from the control. The mean areas under the growth curve and the significant differences identified, aregiven in Table 3, together with the areas expressed as percentages of that of thecontrol. The individual test replicate percentages were transformed to probability scaleand analysed by linear regression against log- concentration, to estimate the median effective concentration (based on biomass curve area, E_bC50) and its 95% confidence limits.

A = (N1-N0)-2No/2*t1+((NI+N2)-2No)/2*(t2-t1)+((Nn-I+Nn)-2No)/2* (tn-tn-1)

where:

No = Cell density at the start of the test (× 10⁴cells/mL)

N1 = Cell density at t1 (× 10⁴cells/mL)

Nn = Cell density at tm (× 10⁴cells/mL)

t1 = Time (days) of first measurement after start of test

t2 = Time (days) of second measurement after start of the test

tn = Time (days) of n^thmeasurement after start of test

The results obtained from these statistical analyses were as follows:

No observed effect (P=0.05) concentration (NOEC) = 2.0 mg/L

Lowest significant effect (P=0.05) concentration = 4.0 mg/L

Median effective concentration, biomass (E_bC5O) = 34 mg/L

95% confidence limits =13 to > 128 mg/L

These data were analysed as described for the area method. The mean growth ratesand the significant differences identified are given in Table 4, together with the rates expressed as percentages of the control. The results obtained from these growth rate analyses expressed on the basis of nominal concentrations were:

No observed effect (P=0.05) concentration (NOEC) = 2 mg/L

Lowest significant effect (P.^,0.05) concentration = 4.0 mg/L

Median effective concentration, growth rate (EC50) = > 128 mg/L

Because the growth rate at each concentration was relatively constant throughout the test period it was not considered necessary to analyse growth rates over shorter intervals.

Tabel 2: Algal cell densities

nominal concentration fo test susbtance	replicate	algal cell density (× 10^4cells/mL)
		Day 1	Day 2	Day 3
control	1.00	6.10	39.60	214.00
	2.00	7.06	50.10	273.00
	3.00	6.94	48.90	254.00
	4.00	6.10	39.40	216.00
	5.00	6.17	42.30	232.00
	6.00	6.20	40.50	224.00
	Mean	6.52	43.50	236.00
1.00	1.00	6.25	38.20	208.00
	2.00	6.27	42.10	229.00
	3.00	6.88	38.30	217.00
	Mean	6.47	39.50	218.00
2.00	1.00	6.15	38.50	208.00
	2.00	6.66	44.50	229.00
	3.00	5.48	33.60	217.00
	Mean	6.10	38.90	218.00
4.00	1.00	5.81	34.10	177.00
	2.00	5.92	36.10	154.00
	3.00	6.36	38.50	147.00
	Mean	5.64	29.40	158.00
8.00	1.00	5.88	31.70	172.00
	2.00	5.42	28.80	154.00
	3.00	5.62	27.80	147.00
	Mean	5.64	29.40	158.00
16.00	1.00	5.04	24.00	128.00
	2.00	5.48	27.50	132.00
	3.00	4.84	243.00	109.00
	Mean	5.12	253.00	123.00
32.00	1.00	5.25	22.80	109.00
	2.00	5.21	23.20	118.00
	3.00	4.87	21.30	102.00
	Mean	5.11	22.40	110.00
64.00	1.00	4.84	20.90	91.80
	2.00	5.31	211.00	101.00
	3.00	4.82	19.90	88.20
	Mean	4.99	21.00	93.70
128.00	1.00	4.83	18.10	72.70
	2.00	4.82	19.00	84.00
	3.00	4.76	15.40	63.90
	Mean	4.80	17.50	73.50

Table 3: Mean areas under the growth curve and percentages of the control indicating statistically significant differences.

Nominal concentration of test substance (mg/L)	Mean area under growth curve (0-72 hours)	% of control
Control	165.2	-
1.00	152.40	92.00
2.00	143.70	87.00
4.00	134.7*	82.00
8.00	111.3*	67.00
16.00	89.3*	54.00
32.00	79.8*	48.00
64.00	70.2*	43.00
128.00	56.5*	34.00

*Significant difference from the control (P=0.05)

Table 4: Mean growth rates over the test period, and percentages of the  control indicating statistically significant differences

Nominal concentration of test substance (mg/L)	Mean growth rate (0-72 hours)	% of control
Control	1.811	-
1	1.785	99
2	1.761	97
4	1.739*	96
8	1.677*	93
16	1.594*	88
32	1.556*	86
64	1.503*	83
128	1.423*	79

Validity criteria fulfilled:

not specified

Conclusions:

Under the study conditions, the nominal 72 h EC50 of the substance based on growth rate and biomass in Selenastrum capricornururn were determined to be greater than 128 mg/L and 34 mg/L respectively (Comber, 1993)

Executive summary:

A study was conducted to determine the toxicity of the substance to the algae Selenastrum capricornutum according to OECD Guideline 201 and EU Method C.3 in compliance with GLP. Algae were exposed to the test substance for 72 h at 1.0, 2.0, 4.0, 8.0, 16.0, 32.0, 64.0 and 128.0 mg/L in a static system. Biomass and growth rate were determined periodically from the test samples. Under the study conditions, the nominal 72 h EC50 of the substance based on growth rate and biomass in Selenastrum capricornutum were determined to be greater than 128 mg/L and 34 mg/L respectively (Comber, 1993).

Key value for chemical safety assessment

EC50 for freshwater algae:

128 mg/L

EC10 or NOEC for freshwater algae:

2 mg/L

Additional information

A study was conducted to determine the toxicity of the substance to the algae Selenastrum capricornutum according to OECD Guideline 201 and EU Method C.3 in compliance with GLP. Algae were exposed to the test substance for 72 h at 1.0, 2.0, 4.0, 8.0, 16.0, 32.0, 64.0 and 128.0 mg/L in a static system. Biomass and growth rate were determined periodically from the test samples. Under the study conditions, the nominal 72 h EC50 of the substance based on growth rate and biomass in Selenastrum capricornutum were determined to be greater than 128 mg/L and 34 mg/L respectively (Comber, 1993).