2,7-Naphthalenedisulfonic acid, 4-amino-5-hydroxy-, coupled with 3-aminophenol, diazotized 5-amino-2-[(4-aminophenyl)amino]benzenesulfonic acid and diazotized benzenamine, 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:

January from 14 to 18, 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

adopted June 7th, 1984.

Qualifier:

according to guideline

Guideline:

other: Draft International Standard ISO/TC 147 (Water quality -Algal growth inhibition test)

Version / remarks:

submitted June 25th, 1987

Principles of method if other than guideline:

The study was performed in compliance with Chemikaliengesetz (Chemical Act) of the Federal Republic of Germany, Anlage 1 (Annex 1), dated March 14th, 1990.

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Analyses of the test substance were performed in duplicate in a sample of the stock solution and of test media of the nominal test concentrations 1.0, 10.0 and 100.0 mg test substance/l and the control: a part of the freshly prepared stock solution and test media (without algae) were sampled and analysed.
For the determination of the stability of the test substance under test conditions, a part of the test media were incubated under the same conditions as in the test itself (but without algae) and analysed after the end of the test (96 hours).
All samples are kept stored at -20 °C to enable additional analyses.

Details on test solutions:

A stock solution was prepared by dissolving the test substance in test water (1.0 g/l). Adequate amounts of the stock solution were added to test water to prepare the tested nominal concentrations.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Scenedesmus subspicatus CHODAT.
- Strain: no 8681 SAG.
- Source: Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der Universität Göttingen.
- Breeding: the algae were grown in the testing laboratories, under standardized conditions according to the OECD Guideline No. 201.
- Preparation of culture medium: prepared with the following final concentrations. Macro-nutrients: 50.0 mg/l of NaHC03, 18.0 mg/l CaCl2.2 H20, 15.0 mg/l NH4CI, 15.0 mg/l MgS04.7 H20, 12.0 mg/l MgCl2.6 H20, 1.6 mg/l KH2PO4. and trace elements: 100.0 μ/l Na2EDTA .2 H20, 80.0 μg/l FeCl3.6 H20, 415.0 μg/l MnCl2.4H2O, 185 μg/l H3BO3, 7.0 μg/l Na2MoO4.2H2O, 3.0 μg/l ZnCl, 1.5 μg/l CoCl2.6H2O and 0.01 μg/l CuCl2.2H2O.
- Other: the cells were taken from a sterile pre-culture, which was set up 72 hours prior to the test at the same conditions as in the test.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

20.3 - 21.7

pH:

Control: 7.8 (start) - 9.6 (end)
Vessels: 7.7 (start) - 10.3 (end)

Nominal and measured concentrations:

Nominal: 1.0, 3.2, 10.0, 32.0 and 100.0 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 ml - Erlenmeyer flasks stoppered with permeable plugs.
- Test solution: 150 ml consisting of sterile nutrient solution, dissolved test substance and algae suspension.
- Initial biomass: 10000 cells/ml test solution.
- No. of vessels per concentration: 3 replicates.
- No. of vessels per control: 2 replicates.

TEST MEDIUM
- Preparation of test medium: prepared with the following final concentrations. Macro-nutrients: 50.0 mg/l of NaHC03, 18.0 mg/l CaCl2.2 H20, 15.0 mg/l NH4CI, 15.0 mg/l MgS04.7 H20, 12.0 mg/l MgCl2.6 H20, 1.6 mg/l KH2PO4. and trace elements: 100.0 μ/l Na2EDTA .2 H20, 80.0 μg/l FeCl3.6 H20, 415.0 μg/l MnCl2.4H2O, 185 μg/l H3BO3, 7.0 μg/l Na2MoO4.2H2O, 3.0 μg/l ZnCl, 1.5 μg/l CoCl2.6H2O and 0.01 μg/l CuCl2.2H2O.
- Preparation of dilution water: same as cultivation medium.

OTHER TEST CONDITIONS
- Sterile test conditions: sterile nutrient solution was used.
- Photoperiod: continuous illumination.
- Light intensity and quality: illumination at about 8000 Lux (measured range: 6720-7890) achieved by fluorescent tubes (universal white L25, 36 W), installed above the shaker in a distance of about 40 cm from the algae flasks.
- Other: room was air-conditioned.

EFFECT PARAMETERS MEASURED: for the determination of cell concentrations, samples of 1 ml test solution were sterily taken out of all flasks after 24, 48, 72 and 96 hours of exposure and not replaced. The algae cell densities in the samples were determinced by counting with an electronical partical counter (Al Cellcounter Model 871).
Inhibition of algae growth was determined from: a) the are under the growth curves A (=biomass); b) the specific growth rates μ for exponentially growing cultures.
The test concentrations corresponding to 50 % inhibition of algae biomass (EbC50) respectively growth rate (EμC50) and their 95 % confidence limits were calculated by the Moving Average Interpolation.

PARAMETERS MEASURED: pH values of test solutions were measured in samples of all test concentrations and controls at the start and at the end of the test. During test duration the temperature was measured daily in an Erlenmeyer flask, filled with water and incubated at the same conditions as the test flasks.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate p.A. (periodically tested - twice a year)

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

17.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

8.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC0

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC0

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

In the control the cell density has increased from nominal N. = 1 x 10^4 cells/ml at the start of the test (0 hours) to N = 127.8 x 10^4 cells/ml (mean value) after 7 2 hours by a factor of about 128.
In the two lowest test concentrations (1.0 and 3.2 mg/l) the algae growth was strongly promoted in relation to the control, but according to the definition of the OECD Guideline No. 201 growth promotion is no adverse effect. Therefore the EC 0 (=NOEC: highest concentration tested without toxic effect) was determined to be 3.2 mg test substance/l.
Test item has obviously an inhibition effect on the growth of Scenedesmus subspicatus first on the concentration of 10.0 mg test substance/l (= LOEC: lowest concentration tested with toxic effect).
In the two highest concentrations (32 and 100 mg/l) the algae growth was strongly inhibited. After 72 hours of exposure to the test substance the algae cell densities seemed to have decreased below the initial densities at the start of the test. This may be caused by a conglomeration of the algal cells-, so that the total cell densities were underestimated by the countings with the particle counter.
The inhibition rate was calculated for both parameter, the algae biomass (determined as the area under the growth curve) and the growth rate μ. Since in the control no further exponential growth was observed after 72 to 96 hours, the growth inhibition was calculated after 72 and 96 hours test duration, but in the following only the results after 72 hours test duration are reported:
Biomass:
- EbC50 (0 - 72 h) 8.4 mg test substance/l (95 % conf. limits not calculable)
- EbC 0 (0 - 72 h) 3.2 mg test substance/l
- EbC100 (0 - 72 h) > 100.0 mg test substance/l
Growth rate:
- ErC 50 (0 - 72 h) 17.4 mg test substance/l (95 % conf. limits: 16.4 - 18.3 mg test substance/l)
- ErC 0 (0 - 72 h): 3.2 mg test substance/l
- ErC100 (0 - 72 h) ≥ 100.0 mg test substance/l

pH CONDITIONS
At the start of the test, the pH-values in the test media were in the range of pH 7.7 to 7.8, at the end of the test pH-values were measured between pH 8.2 and 10.3. The high pH-values in the control and the two lowest test concentrations were obviously caused by the consumption of CO2 by algae during their rapid growth respectively their high densities (although the test flasks have been intensively shaken).

MEASURED CONCETRATION
The test substance concentration in the analysed stock solution amounted to 105.0 % of the nominal value. In the analysed test media of the nominal concentrations 10 and 100 mg/l, test substance concentrations were measured corresponding to between 85.3 and 99.5 % of the nominal values. In the lowest concentration of 1.0 mg/l, the test substance concentration decreased from 87 % of the nominal value at the start of the test to 56 % after 96 hours of exposure.
Since this test concentration was not relevant to the reported results (due to the growth promotion in this concentration) and in the higher test concentrations, the test substance was sufficiently stable under test conditions, the reported results are related to the nominal concentrations of the test substance.

Results with reference substance (positive control):

No results with reference substance because the reference substance is periodically tested - twice a year, but not tested in the present study.

The influence of the test substance on algae growth during the test duration of 96 hours is shown in the following table.

Concentration	Flask no	Cell number (* 10000/ml)
		24 h			48 h			72 h			96 h
control	1	10.8	10.9	10.9	53.3	53.5	53.5	129.7	129.0	130.0	192.0	192.0	195.0
	2	11.4	11.5	11.5	42.0	41.8	41.9	120.7	121.1	10.9	195.0	190.0	190.0
	3	9.9	10.1	10.1	35.6	35.9	37.7	125.7	125.6	125.0	188.0	183.0	185.0
	4	7.9	8.1	8.0	38.7	38.9	38.9	129.9	130.7	130.8	203.0	203.0	203.0
	5	7.4	7.3	7.3	56.8	56.3	56.4	129.4	129.3	130.0	177.0	176.0	176.0
	6	6.8	6.8	6.8	45.1	44.5	44.5	130.5	131.3	130.1	192.0	192.0	192.0
	m	9.077			45.294			127.761			190.222
	s	1.839			7.580			3.647			8.454
1 mg/l	7	8.5	8.9	8.7	75.4	75.5	75.5	312.0	307.0	315.0	371.0	370.0	370.0
	8	8.9	8.9	8.9	67.1	67.3	67.3	301.0	299.0	312.0	369.0	368.0	368.0
	9	6.5	6.6	6.6	73.1	72.7	72.9	253.0	261.0	258.0	367.0	368.0	368.0
	m	8.056			71.867			290.889			368.778
	s	1.125			3.650			25.766			1.302
3.2 mg/l	10	8.1	8.2	8.2	40.4	39.8	40.0	195.0	199.0	194.0	373.0	368.0	368.0
	11	7.2	7.0	7.1	30.5	31.0	31.0	144.0	143.0	144.0	276.0	279.0	278.0
	12	6.8	6.9	6.9	39.0	38.8	39.0	138.0	141.0	145.0	298.0	295.0	295.0
	m	7.378			36.611			160.333			314.444
	s	0.604			4.366			26.861			42.211
10 mg/l	13	2.8	2.7	2.7	5.6	5.6	5.6	15.4	15.1	15.2	48.0	48.0	48.0
	14	2.7	2.6	2.7	4.7	4.4	4.5	8.9	9.2	9.3	30.0	29.0	31.0
	15	2.6	2.6	2.6	4.6	4.6	4.6	4.0	4.0	4.0	33.0	31.0	31.0
	m	2.667			4.911			9.456			36.556
	s	0.0701			0.523			4.872			8.647
32 mg/l	16	3.2	3.3	3.3	4.6	4.6	4.5	6.6	6.8	7.6	1.5	1.5	1.5
	17	2.7	2.7	2.7	3.7	3.7	3.7	5.0	4.9	4.8	1.1	1.1	1.0
	18	2.4	2.5	2.5	3.8	3.8	3.8	4.4	4.4	4.4	1.0	0.9	0.9
	m	2.811			4.022			5.433			1.167
	s	0.359			0.412			1.225			0.260
100 mg/l	19	2.7	2.7	2.2	3.7	3.6	3.6	4.5	4.5	4.3	0.8	0.8	0.8
	20	2.7	2.7	2.7	3.3	3.3	3.3	3.5	3.6	3.4	0.6	0.6	0.6
	21	2.4	2.5	2.5	3.6	3.6	3.6	3.9	3.9	4.8	0.6	0.6	0.6
	m	2.567			3.511			4.044			0.667
	s	0.180			0.162			0.500			0.100

m = arithmetic mean, s = standard deviation

The inhibition rate was calculated for both parameters, the algae biomass (determined as the area under the growth curve) and the growth rate. Since in the control no further exponential growth was observed after 72 to 96 hours, the growth inhibition was calculated after 72 and 96 hours test duration.

Influence of the test substance on algae growth (area under growth curve A and percentage inhibition of A)

	Areas (A) under the growth curve and % inhibition (%)
Concentration (mg/l)	24 h		48 h		72 h		96 h
	A	%	A	%	A	%	A	%
control	97	0.0	725	0.0	2778	0.0	6570	0.0
1.0	85	12.6	1020	-40.6	5349	-92.5	13241	-101.5
3.2	77	21.0	580	20.0	2920	-5.1	8593	-30.8
10.0	20	79.4	87	88.0	235	91.5	763	88.4
32.0	22	77.6	80	89.0	169	93.9	224	96.6
100.0	19	80.6	68	90.7	134	95.2	167	97.5

Influence of the test susbstance on algae growth (growth rate and percentage inhibition)

Nominal test concentration (mg/l)	0 to 72 h		0 to 96 h
	μ	Ιμ (%)	μ	Ιμ (%)
control	1.617	-	1.313	-
1.0	1.893	-17.1	1.478	-12.6
3.2	1.693	-4.7	1.438	-9.5
10.0	0.750	53.6	0.900	31.4
32.0	0.563	65.2	0.040	97.0
100.0	0.467	71.1	-0.100	107.6

Iμ = percentage inhibition growth rate

Negative values stand for % growth promotion, not inhibition.

pH and temperature values in untreated and treated test media at the start and the end of the test.

Nominal test concentration (mg/l)	pH-values
	Start	End
control	7.8	9.6
1.0	7.7	10.3
3.2	7.7	9.5
10.0	7.7	8.9
32.0	7.7	8.5
100.0	7.7	8.2
	Temperature (°C)
day 0 (start)	20.3 *
day 1	21.7
day 2	21.4
day 3	21.0
day 4 (end)	21.0

* measured in the freshly prepared test medium.

The high pH-values in the control and the two lowest test concentrations were obviously caused by the consumption of CO₂ by algae during their rapid growth respectively theri high densities (although the test flasks were intensively shaken).

Measured concentrations

Nominal conc. (mg/l)	Sampling period (d)	Conc. of test item found (mg/l)*	% of nominal
1.0	0	0.87	87.0
1.0	4	0.56	56.0
10.0	0	9.83	98.3
10.0	4	8.53	85.3
100.0	0	99.50	99.5
100.0	4	94.80	94.8
1000.00 (stock solution)	0	1050.00	105.0
Control sample	0	--- **	---
Control sample	4	--- **	---

* Mean value of a repeated test

** Counts were below lowest calibration point

Validity criteria fulfilled:

yes

Remarks:

biomass concentration in the control cultures was increased by a factor of at least 16 within 3 days

Conclusions:

ErC50 (72 h): 17.4 mg/l (nominal)
EbC50 (72 h): 8.4 mg/l (nominal)

Executive summary:

The influence of test item on the growth of the green alga Scenedesmus subspicatus Chodat was investigated in a 96-hour static test according to the OECD Guideline No. 201, adopted June 7, 1984 and the Draft ISO/TC 147, submitted June 25, 1987.

The nominal concentrations tested were 1.0, 3.2, 10.0, 32.0 and 100.0 mg test substance/l and a control without any additions. Test item has an inhibition effect on the growth of Scenedesmus subspicatus first on the concentration of 10.0 mg test substance/l (= LOEC: lowest concentration tested with toxic effect). The EC 0 (= NOEC: highest concentration tested without toxic effect) was determined to be 3.2 mg test substance/l.

The inhibition rate was calculated for both parameter, the algae biomass (determined as the area under the growth curve) and the growth rate. Since in the control no further exponential growth was observed after 72 to 96 hours, only the results after 72 hours test duration are reported:

Biomass:

- EbC50 (0 - 72 h) 8.4 mg test substance/l (95 % conf. limits not calculable)

- EbC 0 (0 - 72 h) 3.2 mg test substance/l

- EbC100 (0 - 72 h) > 100.0 mg test substance/l

Growth rate:

- ErC 50 (0 - 72 h) 17.4 mg test substance/l (95 % conf. limits: 16.4 - 18.3 mg test substance/l)

- ErC 0 (0 - 72 h): 3.2 mg test substance/l

- ErC100 (0 - 72 h) ≥ 100.0 mg test substance/l

The test substance concentration in the analysed stock solution amounted to 105.0 % of the nominal value, in the analysed test media of the nominal concentrations 10 and 100 mg/l, test substance concentrations were measured corresponding to between 85.3 and 99.5 % of the nominal values. In the lowest concentration of 1.0 mg/l, the test substance concentration decreased from 87 % of the nominal value at the start of the test to 56 % after 96 hours of exposure. Since this test concentration was not relevant to the reported results and in the higher test concentrations, the test substance was sufficiently stable under test conditions, the reported results are related to the nominal concentrations of the test substance.

Conclusion

ErC50 (72 h): 17.4 mg/l (nominal)

EbC50 (72 h): 8.4 mg/l (nominal)

Description of key information

ErC50 (72 h): 17.4 mg/l (nominal)

EbC50 (72 h): 8.4 mg/l (nominal)

Key value for chemical safety assessment

EC50 for freshwater algae:

17.4 mg/L

EC10 or NOEC for freshwater algae:

3.2 mg/L

Additional information

The influence of test item on the growth of the green alga Scenedesmus subspicatus was investigated in a 96-hour static test, according to the OECD Guideline No. 201. The nominal concentrations tested were 1.0, 3.2, 10.0, 32.0 and 100.0 mg test substance/l and a control without any additions. Test item has an inhibition effect on the growth of Scenedesmus subspicatus first on the concentration of 10.0 mg test substance/l; the EC0 was determined to be 3.2 mg test substance/l. The EbC50 at 72 hours resulted to be 8.4 mg test substance/l, while the ErC 50 (72 h) was indicated to be 17.4 mg test substance/l. The test substance concentration in the analysed stock solution amounted to 105.0 % of the nominal value, in the analysed test media of the nominal concentrations 10 and 100 mg/l, test substance concentrations were measured corresponding to between 85.3 and 99.5 % of the nominal values. In the lowest concentration of 1.0 mg/l, the test substance concentration decreased from 87 % of the nominal value at the start of the test to 56 % after 96 hours of exposure. Since this test concentration was not relevant to the reported results and in the higher test concentrations, the test substance was sufficiently stable under test conditions, the reported results are related to the nominal concentrations of the test substance).

A second test on the substance under registration is available. The effects of the test item on the growth of a green algae species, Pseudokirchneriella subcapitata, was tested in a 72 - hour test according to the OECD guideline 201. Six different concentrations of the substance, ranging from 1 - 48 mg/l, and a control, without test addition, were in contact for 72 hour with the algae. The cells were counted after 72 hours of exposure. The growth inhibition percentage was estimated by the difference between the biomass in the start and the biomass at the end of the test. pH was measured at the test initiation and at the end. The EC50, after 72 hours, resulted to be 10 mg/l (nominal), based on active ingredient.