Tetrasodium 4,4'-bis[[4-morpholino-6-(p-sulphonatoanilino)-1,3,5-triazin-2-yl]amino]stilbene-2,2'-disulphonate

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:

Experiment start date - 27 June 2008; Experiment completion date - 07 August 2008; Study completion date - 31 October 2008.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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 (incl. QA statement)

Specific details on test material used for the study:

Identity: FAT 66042/A
Batch No.: 0582900
Purity: >80 %
Expiration date: 25-Feb-2013
pH: 9.5 at concentration of 10 g/L
Aggregate state / physical form at room temperature: solid
Storage conditions: At room temperature at about 20 °C, away from direct sunlight.

Analytical monitoring:

yes

Details on sampling:

A sample was taken from the control and from the nominal test concentration of 100 mg/L.

Vehicle:

no

Details on test solutions:

The test medium of the highest nominal concentration of 100 mg/L was prepared by dissolving 50.3 mg of the test item completely in 500 mL of test water using ultrasonic treatment for 15 minutes and intense stirring for 15 minutes at room temperature. The test medium of the highest test concentration was diluted with test water to prepare the test media of the lower test concentrations. The test media were prepared just before the start of the test.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum).
- Strain: strain No. 61.81 SAG.
- Source: Supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, Göttingen / Germany). The algae were cultivated in RCC' s laboratories under standardized conditions according to the test guidelines.
- Age of inoculum: An inoculum culture was set up four days before the start of the test.
- Method of cultivation: The algae were cultivated under the test conditions.

CONTROL
For evaluation of the algal quality and experimental conditions, potassium dichromate is tested as a positive control twice a year to demonstrate satisfactory test conditions. The result of the latest positive control test performed in 2008 showed that the sensitivity of the test system was within the historical range of the laboratory (72-hour EC50 for the growth rate: 1.20 mg/l, range of the 72-hour EC50 for the growth rate from 2000 to 2008: 0.71-1.74 mg/l).

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

The water temperature during the test was between 22 and 23 °C; the water temperature was measured and recorded daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks.

pH:

At the start of the test, the pH measured in the treatments was between 7.9 and 8.0. At the end of the test, pH values between 8.0 and 8.4 were measured. The increase of the pH during the test was caused by the uptake of CO2 by the algae due to their growth.

Nominal and measured concentrations:

0, 4.6, 10, 22, 46, 100 mg/L

Details on test conditions:

TEST SYSTEM
- Inoculum: The inoculum culture was diluted threefold one day before the start of the test to ensure that the algae were in the exponential growth phase when used to inoculate the test solutions.
- Test vessel: 50 ml Erlenmeyer flasks were used per replicate containing 15 ml of test solution.
- Type: Each test flask was covered with a glass dish.
- Stirring: During the test, the test solutions were continuously stirred by magnetic stirrers.

TEST MEDIUM / WATER PARAMETERS
- Test water: reconstituted test water prepared according to the test guidelines was used for algal cultivation and testing. Analytical grade salts were dissolved in sterile purified water to obtain the following concentrations:
Macro-nutrients
NaHCO3: 50.0 mg/l
KH2PO4: 1.6 mg/l
MgSO4 x 7 H2O: 15.0 mg/l
MgCl2 x 6 H2O: 12.0 mg/l
CaCl2 x 2 H2O: 18.0 mg/l
NH4Cl: 15.0 mg/l
Trace elements
H3BO3: 185.0 µg/l
MnCl2 x 4 H2O: 415.0 µg/l
ZnCl2: 3.0 µg/l
CoCl2 x 6 H2O: 1.5 µg/l
CuCl2 x 2 H2O: 0.01 µg/l
Na2MoO4 x 2 H2O: 7.0 µg/l
FeCl3 x 6 H2O: 64.0 µg/l
Na2EDTA x 2 H2O: 100.0 µg/l
The water hardness (calculated) of the test water was 0.24 mmol/l (= 24 mg/l as CaCO3).

OTHER TEST CONDITIONS
- Photoperiod: The test flasks were illuminated by fluorescent tubes (Philips TLD 36W/840), installed above the test flasks.
- Light intensity and quality: Approximately 7400 Lux (range: 6370 to 8000 Lux, measured at nine places in the experimental area).

EFFECT PARAMETERS MEASURED
- Determination of Algal Biomass: A small volume of the algal suspension was daily withdrawn from each test flask for the measurement of the biomass, and was not replaced. The algal biomass in the samples was determined by measurement of the algal cell density using an electronic particle counter (Coulter Counter®, Model ZM). The measurements were performed at least in duplicate. At the end of the test, a sample was taken from the control and from the nominal test concentration of 100 mg/l. The shape and size of the algal cells were visually inspected.
- Determination of Algal Growth Inhibition and EC Values: Growth rate and yield were calculated for each test flask. The mean values for growth rate and yield were calculated for each treatment. The EC10 for the inhibition of yield were calculated by Probit Analysis.

TEST CONCENTRATIONS
- Test medium: Test medium of the highest nominal concentration of 100 mg/l was prepared by dissolving 50.3 mg of the test item completely in 500 ml of test water using ultrasonic treatment for 15 minutes and intense stirring for 15 minutes at room temperature. The test medium of the highest test concentration was diluted with test water to prepare the test media of the lower test concentrations. The test media were prepared just before the start of the test.
- Range finding study: Yes

RANGE FINDING TEST
The selection of the test concentrations was based on the results of a range-finding test and on results of a pre-experiment to determine the water solubility of the test item in the test water (non-GLP). The following nominal concentrations of the test item were tested: 4.6, 10, 22, 46 and 100 mg/l. Additionally, a control was tested in parallel (test water without test item). The test design included three replicates per test concentration and six replicates of the control. The test was started using a nominal algal cell density of 10000 cells/ml. The initial cell density was selected according to the recommendations of the OECD test guideline. The algal cell density in the pre-culture was determined by an electronic particle counter (Coulter Counter®, ModelZM). A static test design was applied. The duration of the test was 72 hours.

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

22 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

46 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

22 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

46 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

The measured concentrations of the test item in the test media of the test concentrations of 22 to 100 mg/l were between 95 and 104 % of the nominal values at the start and the end of the test. The test item was stable in the test media over the test period of 72 hours. The biological results were related to the nominal concentrations of the test item. The test item had a significant inhibitory effect on the growth rate and yield after the test period of 72 hours at the concentrations of 46 and 100 mg/l (results of Dunnett's tests, one-sided, a = 0.05). Thus, the concentration of 46 mg/l was determined to be the 72-hour LOEC. The 72-hour NOEC was determined to be 22 mg/l, since up to and including this test concentration the growth rate and yield of the algae after 72 hours were not significantly lower than in the control. The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the test concentration of 100 mg/L and the algal cells in the control. The shape and size of the algal cells were obviously not affected by the test item up to at least this concentration.

The biological results can be summarized as follows:

Parameter (0-72 h)	Growth rate	Yield
EC10 (mg/l)	>100	46
95 % confidence interval	nd	nd
EC20 (mg/l)	>100	>100
95 % confidence interval	nd	nd
EC50 (mg/l)	>100	>100
95 % confidence interval	nd	nd
NOEC (mg/l)	22	22
LOEC (mg/l)	46	46

n.d. could not be determined

In the control the biomass increased by a factor of 111 over 72 hours. The validity criterion of increase of biomass by at least a factor of 16 within three days was fulfilled. The mean coefficient of variation of the daily growth rates in the control (section-by-section growth rates) during 72 hours was 17 %. According to the OECD test guideline, the mean coefficient of variation must not be higher than 35 %. Thus, the validity criterion was fulfilled. The coefficient of variation of the average specific growth rates in the replicates of the control after 72 hours was 1.9 %. According to the OECD test guideline, the coefficient of variation must not be higher than 7 %. Thus, the validity criterion was fulfilled. No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test period.

Biomass of Algae

Nominal test item concentration (mg/l)	Rep. N.	Biomass of algae* (algal cell density in Nx10^4 cells/ml)
		24 hrs	48 hrs	72 hrs
Control	1	4	26.1	119.6
	2	4.2	27.3	117.1
	3	4.1	26.4	98.7
	4	4.1	26.4	119
	5	4.2	26.5	112.4
	6	4.3	27	99.3
	Mean	4.1	26.6	111
	SD	0.1	0.4	9.6
4.6	1	3.9	25.5	112.2
	2	4.8	27.9	121.6
	3	4	27.1	117.6
	Mean	4.2	26.8	117.1
	SD	0.5	1.2	4.7
10	1	4.5	26.7	125.7
	2	4.4	26.9	115.5
	3	4.4	26.3	118.7
	Mean	4.4	26.6	120
	SD	0.1	0.3	5.2
22	1	4.2	27.4	115
	2	4.4	28.9	99.4
	3	4.1	26.8	105.4
	Mean	4.2	27.7	106.6
	SD	0.2	1.1	7.9
46	1	4.2	30.1	97.4
	2	4.3	29.3	86.8
	3	4.2	28.2	99
	Mean	4.2	29.2	94.4
	SD	0.1	0.9	6.6
100	1	4.2	29.8	100.9
	2	4.4	30.3	87.7
	3	4.5	30.3	94.3
	Mean	4.3	30.1	94.3
	SD	1	0.3	6.6

SD: Standard deviation

*. The biomass was determined by cell counting (at least duplicate measurements per replicate). At the start of the test, the initial cell density was 1 x 10⁴ algal cells/ml).

Average Growth Rates (µ)

Nominal test item concentration (mg/l)	Average growth rate µ/day and inhibition of µ (Ir)
	0-24 hrs		0-48 hrs		0-72 hrs
	µ	Ir (%)	µ	Ir (%)	µ	Ir (%)
Control	1.42	0.0	1.64	0.0	1.57	0.0
4.6	1.44	-1.5	1.64	-0.2	1.59	-1.2
10	1.48	-4.6	1.64	0.0	1.60	-1.7
22	1.43	-1.2	1.66	-1.2	1.56	0.8
46	1.44	-1.6	1.69	-2.8	1.52*	3.4
100	1.47	-3.5	1.70	-3.8	1.51*	3.4

*: mean value significantly lower than in the control (according to a Dunnett's-test, one-sided, α = 0.05)

Yield (Y)

Nominal test item concentration (mg/l)	Yield Y (x 10^4) and inhibition of Y (Iy)
	0-24 hrs		0-48 hrs		0-72 hrs
	Y	Iy(%)	Y	Iy(%)	Y	Iy(%)
Control	3.1	0.0	25.6	0.0	110.0	0.0
4.6	3.2	-3.5	25.8	-0.9	116.1	-5.6
10	3.4	-8.8	25.6	0.0	119.0	-8.1
22	3.2	-2.4	26.7	-4.3	105.6	4.0
46	3.2	-2.9	28.2	-10.2	93.4*	15.1
100	3.3	-6.7	29.1	-13.8	93.3*	15.2

*: mean value significantly lower than in the control (according to a Dunnett's-test, one-sided, α = 0.05)

Section·-by-Section Growth Rates

Nominal test item concentration (mg/l)	Section-by-section growth rates (day^-l) and inhibition of the growth rates (Ir)
	0-24 hrs		0-48 hrs		0-72 hrs
	µ	Ir(%)	µ	Ir(%)	µ	Ir(%)
Control	1.42	0.0	1.86	0.0	1.43	0.0
4.6	1.44	-1.5	1.85	0.7	1.47	-3.4
10	1.48	-4.6	1.80	3.5	1.51	-5.6
22	1.43	-1.2	1.89	-1.2	1.35	5.6
46	1.44	-1.6	1.93	-3.8	1.17	17.8
100	1.47	-3.5	1.94	-4.1	1.14	20.1

Analytical results;

The measured concentrations of the test item in the test media of the test concentrations of 22 to 100 mg/L were between 95 and 104 % of the nominal values at the start and the end of the test. The test item was stable in the test media over the test period of 72 hours. The R2 fit of the calibration curve used was 0.9997. This reflects the linearity of the HPLC-system within the calibration range of 10.3 - 124 mg test item/L. Concurrent with the sample analysis, recoveries of spiked test water samples at relevant concentrations (101 and 22.2 mg test item/L) were perfonned in duplicate. The average recoveries for the non-centrifuged samples were found to be 106 % and 107 % of the spiked values, with an overall mean of 106 % (n = 4). The average recoveries for the centrifuged samples were found to be 105 % and 106 % of the spiked values, with an overall mean of 106 % (n = 4). No correction for the recovery rate was made. The limit of quantification for the test item in the injected solution (LOQ) was derived from the lowest calibration solution, which fits into the calibration curve: the value is 10.3 mg test item/L. The biological control samples and an analysed analytical blank (test water) did not significantly affect the HPLC-chromatogram at the retention time of the test item. The average recoveries found in the treatment samples at test start ranged from 99 % to 104 % and at test end from 95 % to 101 % of the nominal concentrations.

Validity criteria fulfilled:

yes

Remarks:

The increase of biomass was at least of a factor of 16, the mean coefficient of variation for section-by-section specific growth rates did not exceed 35 % and the coefficient of variation of the average specific growth rates was not higher than 7 %

Conclusions:

The EC50 (72 h) of FAT 66042/A was determined to be greater than 100 mg/L.

Executive summary:

The influence of the test item on the growth of the freshwater green algal species Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum) was investigated in a 72-hour static test according to the OECD Guideline 201 (2006) and the ED Commission Directive 92/69/EEC, C.3 (1992). The nominal concentrations of the test item of 4.6, 10, 22, 46 and 100 mg/l were tested in parallel with a control.

The measured concentrations of the test item in the test media of the test concentrations of 22 to 100 mg/L were between 95 and 104 % of the nominal values at the start and the end of the test. The test item was stable in the test media over the test period of 72 hours. The test item had a significant inhibitory effect on the growth rate and yield after the test period of 72 hours at the concentrations of 46 and 100 mg/l. Thus, the concentration of 46 mg/l was determined to be the 72-hour LOEC. The 72-hour NOEC was determined to be 22 mg/l, since up to and including this test concentration the growth rate and yield of the algae after 72 hours were not significantly lower than in the control.

Description of key information

The influence of the test item on the growth of the freshwater green algal species Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum) was investigated in a 72-hour static test according to the OECD Guideline 201 (2006) and the ED Commission Directive 92/69/EEC, C.3 (1992). The nominal concentrations of the test item of 4.6, 10, 22, 46 and 100 mg/l were tested in parallel with a control.

The measured concentrations of the test item in the test media of the test concentrations of 22 to 100 mg/L were between 95 and 104 % of the nominal values at the start and the end of the test. The test item was stable in the test media over the test period of 72 hours. The test item had a significant inhibitory effect on the growth rate and yield after the test period of 72 hours at the concentrations of 46 and 100 mg/l. Thus, the concentration of 46 mg/l was determined to be the 72-hour LOEC. The 72-hour NOEC was determined to be 22 mg/l, since up to and including this test concentration the growth rate and yield of the algae after 72 hours were not significantly lower than in the control.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

22 mg/L