REAKTIV-GELB F-68 072 FW

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:

14 June 2006 to 28 July 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

yes

Remarks:

To reduce shading effects of the coloured test substance, the test was earned out with strong illumination (110 - 120 uE) and with a reduced test volume of 25 ml in 300 ml Erlenmeyer flasks.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

To reduce shading effects of the coloured test substance, the test was earned out with strong illumination (110 - 120 uE) and with a reduced test volume of 25 ml in 300 ml Erlenmeyer flasks.

Principles of method if other than guideline:

The above deviation is recognised for coloured substances, and is considered appropriate for the testing of dyestuffs.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable.

Analytical monitoring:

yes

Details on sampling:

In order to avoid an impairment of the test system, an additional replicate was used for analysis and pH measurement at the beginning of the test (control: replicate VII; test concentrations: replicate IV). Analysis at the end of the test is performed using replicate I, while pH measurement is performed using the total volume replicates I-III.

In order to check whether or not significant amounts of the test item are incorporated into the algal biomass during the test period, a test flask at the highest test concentration without algae was used in parallel to the geometric series of test concentrations. The results of measured item concentrations in these test media without algae are indicated by the symbol.

Vehicle:

no

Details on test solutions:

A stock solution was prepared to give the desired series of test concentrations. To achieve this 125.0 mg of the test item were added to 1 litre of dilution water.

Analytical Procedure - Standard Solutions used for Calibration
21.2 mg of the test item were dissolved in 50% Acetonitrile (50 ml Acetonitrile and 50 ml Milliporewater) and made up with 50% Acetonitrile to the mark in a 100 ml volumetric flask to prepare a stock solution of 212 mg/1. Defined volumes of this stock solution were diluted with 50% Acetonitrile to obtain standard solutions in the range of 0.053 to 4.24 mg/1.

Although the overall method sensitivity may be better, the concentration of the lowest used calibration solution is employed as the limit of quantification.

These solutions were used to calibrate the HPLC-system.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

Name: Desmodesmus subspicatus
Source: Non-axenic strain of the test species obtained from 'The Collection of Algal Cultures' of the Institute of Plant Physiology at the University of Gottingen (Germany)
Maintenance of stock cultures: Exponentially-growing stock cultures are maintained in the test facility under constant temperature conditions (23 +- 2°C) at a light intensity in the range 60 - 120 μE. x m-2 x s-1 (measured in the range 400 to 700 nm using a spherical quantum flux meter). The nutrient medium (according to BRINGMANN & KUHN (1977) is renewed once a week. Cell density measurements are made using a microcell counter.
Preparation of pre-cultures: Pre-cultures are set up three days before the start of a test. They are grown under identical exposure conditions as the stock cultures, except from the use of a different nutrient medium.
Test cultures: The algal inocula for a test are taken from an exponentially-growing pre-culture and are mixed with the nutrient medium to make up to a final cell density of about 5000 cells per millilitre in the test medium

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No post exposure period

Hardness:

No data

Test temperature:

21-25 deg C

pH:

8

Dissolved oxygen:

No data

Salinity:

Not applicable.

Nominal and measured concentrations:

1.9, 4.3, 9.4, 20.7, 45.5 and 100 mg/l stock solution

Details on test conditions:

Test vessels: 300 ml Erlenmeyer flasks with cotton stoppers
Culturing apparatus: Light chamber in which a temperature in the range 21°C to 25°C can be maintained at +/- 2°C, and continuous uniform illumi nation is provided in the spectral range 400 to 700 nm. Magnetic stirrers kept the algae in suspension.
Light intensity: At the average of the test solutions, a light intensity in the range 110 to 120 μE. x m-2 x s-1 , or an equivalent range of 7333 to 8000 lx, is recommended to use.
Cell density measurements: Cell densities are measured in a microcell counter or, alternatively, are determined by means of a microscopic counting chamber.
Experimental design: 6 test concentrations plus 1 control
3 replicates per concentration, 6 replicates per control
initial cell density in the test cultures approximately 5000 cells per millilitre
additionally highest test concentration without algae
Test concentration/s (nominal): 1.9, 4.3, 9.4, 20.7, 45.5 and 100 mg/1
Method of administration: stock solution
Duration of exposure: 72 hours
Criteria of effects: The criteria of adverse effects used in this study were the item-induced inhibition of growth [b] and growth rate [r], respectively, of the algal population.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

65.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

3.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

908 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

30.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

4.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

20.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Analysis of the growth and growth rate of the algal population within the 72 h exposure period (by probit analysis and DUNNETT's test, respectively) give the following results:

Results [mg/L]:
95% confidence limits [mg/L]
EbC 50 (0-72 h) : 65.6 45.7 - 113
EbC10(0-72 h) : 3.8 1.3 - 6.8
ErC 50 (0-72 h) : 908 496 - 2472
ErC 10 (0-72 h) : 30.9 22.9 - 38.5
NOEC [b] (ta 0.05) : 4.3
NOEC [r] (ta 0.05) : 20.7

All results are expressed in terms of nominal concentrations concentrations. Recovery rates ranged from 85.4 - 89.4% of nominal values at 0 hours, and from 84.7 - 88.6% of nominal values at 72 hours, respectively.

Results with reference substance (positive control):

Not applicable.

Reported statistics and error estimates:

Growth and growth rates were used to calculate a No Observed Effect Concentration and a Lowest Observed Effect Concentration according to DUNNETT (1955, 1964). No significant statistics or error estimates were reported.

 

Mean growth (b) [integral of biomass]

 

Nominal concentration [mg/L]	Area under growth curve	Inhibition (+) / Increase (-) [%]
Control	320278	0.0
1.9	339444	-6.0
4.3	270000	15.7
9.4	247778	22.6
20.7	231111	27.8
45.5	172778	46.1
100	128889	59.8

 

Mean growth rate (r)

 

Nominal concentration	Growth rate	Inhibition (+) / Increase (-)
[mg/L]	[1/d]	[%]
Control	1.50	0.0
1.9	1.51	-0.8
4.3	1.47	2.2
9.4	1.42	5.3
20.7	1.41	6.2
45.5	1.31	13.0
100	1.21	19.4

Validity criteria fulfilled:

yes

Conclusions:

Analysis of the growth and growth rate of the algal population within the 72 h exposure period (by probit analysis and DUNNETT's test, respectively) give the following results:

Results [mg/l]:
95% confidence limits [mg/l]
EbC 50 (0-72 h) : 65.6 45.7 - 113
EbC10(0-72 h): 3.8 1.3 - 6.8
ErC 50 (0-72 h): 908 496 - 2472
ErC 10 (0-72 h): 30.9 22.9 - 38.5
NOEC [b] (ta 0.05): 4.3
NOEC [r] (ta 0.05): 20.7

Executive summary:

A study was performed to assess adverse effects of Reaktiv Gelb F68072 FW on the growth (= increase in cell density) and the growth rate (= rate of increase in cell density with time) of the planktonic freshwater algal species Desmodesmus subspicatus over several generations.

The study was conducted in accordance with EEC Methods for Determination of Ecotoxicity Annex to Directive 92/69/EEC (O.J. No. L383A, 29.12.92) Part C, Method 3 'Algal inhibition test' which is in most parts equivalent to the OECD Guideline for Testing of Chemicals No. 201 'Alga, Growth Inhibition Test'. Study conducted in compliance to with GLP.

 

Exponentially growing algal cells were exposed for a period of 72 hours to a range of concentrations, nominally 1.9, 4.3, 9.4, 20.7, 45.5 and 100 mg/1 of Reaktiv Gelb F68072 FW dissolved in water.

The cell densities were measured at 24 hour intervals. Inhibition of the algal population was measured as reduction in growth (indexb) and growth rate (indexr), relative to control cultures grown under identical conditions. Growth and growth rates were used to calculate a No Observed Effect Concentration and a Lowest Observed Effect Concentration according to DUNNETT (1955, 1964). The following values were determined:

Results [mg/1]: EbC 50 (0-72 h)	: 65.6	95% confidence limits [mg/1] 45.7-113
EbC 10 (0-72 h)	: 3.8	1.3 - 6.8
ErC 50 (0-72 h)	: 908	496 - 2472
ErC 10 (0-72 h)	: 30.9	22.9 - 38.5
NOEC [b] (ta0.05)	: 4.3
NOEC [r] (ta0.05)	: 20.7

 All results are expressed in terms of nominal concentrations. Recovery rates ranged from 85.4 - 89.4% of nominal values at 0 hours, and from 84.7 - 88.6% of nominal values at 72 hours, respectively.

To reduce shading effects of the coloured test substance, the test was earned out with strong illumination (110 - 120 uE) and with a reduced test volume of 25 ml in 300 ml Erlenmeyer flasks.

The test substance is not deemed to trigger the requirements for classification and labelling on the basis of these results alone, as it has:

1. a) Been proven to degrade rapidly in the aquatic environment (on the basis of hydrolysis)


2. b) Has an absence of chronic effects at 1.0 mg/l as demonstrated in the chronic toxicity to Daphnia study.

Description of key information

Under the conditions of the study, the E_r50 (72h) for Desmodesmus subspicatus was found to be 908 mg/L test substance (nominal concentration). Further, the NOEC for growth rate was found to be 20.7 mg/L (nominal) test substance.

Key value for chemical safety assessment

EC50 for freshwater algae:

908 mg/L

EC10 or NOEC for freshwater algae:

20.7 mg/L

Additional information

A study was performed to assess adverse effects of Reaktiv Gelb F68072 FW on the growth (= increase in cell density) and the growth rate (= rate of increase in cell density with time) of the planktonic freshwater algal species Desmodesmus subspicatus over several generations.

The study was conducted in accordance with EEC Methods for Determination of Ecotoxicity Annex to Directive 92/69/EEC (O.J. No. L383A, 29.12.92) Part C, Method 3 'Algal inhibition test' which is in most parts equivalent to the OECD Guideline for Testing of Chemicals No. 201 'Alga, Growth Inhibition Test'. Study conducted in compliance to with GLP.

All results are expressed in terms of nominal concentrations concentrations. Recovery rates ranged from 85.4 - 89.4% of nominal values at 0 hours, and from 84.7 - 88.6% of nominal values at 72 hours, respectively.

To reduce shading effects of the coloured test substance, the test was earned out with strong illumination (110 - 120 uE) and with a reduced test volume of 25 ml in 300 ml Erlenmeyer flasks.

The test substance is not deemed to trigger the requirements for classification and labelling on the basis of these results alone, as it has:

1. a) Been proven to degrade rapidly in the aquatic environment (on the basis of hydrolysis)


2. b) Has an absence of chronic effects at 1.0 mg/l as demonstrated in the chronic toxicity to Daphnia study.