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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
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:
no
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Batch number: CN61204501
Date of receipt: 2017-03-02
Expiry date: 2018-07-24
Purity: insoluble
Stability of test concentration/s during exposure: Examined by chemical analysis (Zinc) at 0 and 72 hours.

Analytical monitoring:
yes
Details on sampling:
Stability of test concentration/s during exposure: Examined by chemical analysis (Zinc) at 0 and 72 hours.
Vehicle:
no
Details on test solutions:
Pre-treatment of test item and preparation of test item concentrations
A stock solution was prepared to give the desired series of test concentrations. 199.9 mg of the test item were added to 2 litres of dilution water, treated for 1 h in an ultrasonic bath and stirred for 24 h on a magnetic stirrer. Undissolved particles of the test item were removed by filtration using a folded filter with a pore size of 7 - 12 µm and an aseptic filter Sartobran sterile capsules with a pore size of 0.45 µm + 0.2 µm. The pH was measured to be 7.9.
To produce the different test item concentrations appropriate amounts of the stock solution were diluted with dilution water to a volume of 100 mL and 0.199 mL of the algal inoculum was added to each replicate resulting in a final cell density of 5000 cells/mL. For each test item concentration and the control 3 replicates were prepared. All flasks were sealed with cotton stoppers.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
Name: Desmodesmus subspicatus (formerly Scene-desmus subspicatus) Strain No. 86.81 SAG
Source: Strain of the test species obtained from 'The Collection of Algal Cultures' of the Institute of Plant Physiology at the University of Göttingen (Germany).
Maintenance and Acclimatisation: Exponentially growing stock cultures were maintained in the test facility under constant temperature conditions (21 - 24 °C with a maximum fluctuation of +/- 2 °C) at a light intensity in the range 60 to 120 µE x m-2 x s-1 (measured in the range 400 to 700 nm using a spherical quantum flux meter). The growth medium (according to BRINGMANN & KÜHN (1977) was renewed once a week. Cell density measurements were made using a microcell counter, Sysmex F300, Digitana.
Preparation of pre cultures: Pre cultures were set up three days before the start of a test. They were grown under identical exposure conditions as the stock cultures, except from the use of a different growth medium.
Test cultures: The algal inocula for the test were taken from an exponentially growing pre culture and were mixed with the growth medium to make up to a final cell density of about 5000 cells per millilitre in the test medium.
Growth medium and dilution water: Growth medium (OECD medium of OECD TG 201, annex 1) was used for the growth of the algae in the pre cultures and the preparation of stock and test solutions of the test item.
Test type:
not specified
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
21 - 24 °C
pH:
7.9 - 8.7
Nominal and measured concentrations:
nominal: 4.3, 9.4, 20.7, 45.5, 100 mg/L
Details on test conditions:
EXPOSURE CONDITIONS
Test vessels: 300 mL Erlenmeyer flasks with cotton stoppers, test volume: 100 mL
Culturing apparatus: Shaking incubator in which a temperature in the range 21 °C to 24 °C was maintained at +/- 2 °C, and continuous uniform illumination was provided in the spectral range 400 to 700 nm. Temperature was measured and recorded daily.
Light intensity: A light intensity ranging from 60 to 120 µE x 1/(m2 x s), or an equivalent range of 4000 to 8000 lux, was measured. The light intensity was checked before the start of the study.
Cell density measurements: Cell densities were measured in a microcell counter (Particle Count and Size Analyzer Z2, Beckman Coulter) by taking aliquots from each test flask.
Experimental design: 5 concentrations plus 1 control; 3 replicates per concentration, 3 replicates per control; Initial cell density in the test cultures appro-ximately 5000 cells per millilitre; Additionally highest test concentration without algae.
Test item concentration/s: 4.3, 9.4, 20.7, 45.5 and 100 mg/L
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 yield [y] and growth rate [r] of the algal population.



Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 4.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: A statistical evaluation of the EC data is not possible
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 4.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: A statistical evaluation of the EC data is not possible
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1.7 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
4.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 4.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: A statistical evaluation of the EC data is not possible
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 1.7 - < 4.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: A statistical evaluation of the EC data is not possible
Details on results:
A statistical evaluation of the EC data is not possible.
The content of the test item during the exposure period was verified by Zinc determination.
The results are expressed in terms geometric mean measured concentrations. The results refer to the test item concentrations calculated on base of the mean measured Zinc values. According to the product information
1 mg/L Zinc equals to 7.01 mg/L test item (molecular weight 458.0 g/mol, empirical formula C18H20N2S4Zn).
Effective concentrations ranged from 4.3 % to 5.0 % of nominal values at 0 hours, and from 1.6 % to 4.0 % of nominal values at 72 hours.
NOEC and LOEC determined according to Williams Multiple Sequential t-test.
Reported statistics and error estimates:
All calculations were carried out using the statistics programme ToxRatPro Version 2.10 (released 2010-09-10). For the calculations all algae counts were divided by a factor of 10000.
Validity criteria fulfilled:
yes
Conclusions:
A study to investigate adverse effects of Vulkacit P Extra N on the growth rate (and yield) of a population of the planktonic freshwater algal species Desmodesmus subspicatus was carried out showing an ErC50 > 4.3 mg/L and a NOEC of 1.7 mg/L.
Executive summary:

A study was performed to assess the adverse effects of Vulkacit P Extra N on the growth rate (= rate of increase in cell density with time) and the yield (= biomass at time t minus initial biomass) of the planktonic freshwater algal species Desmodesmus subspicatus (former name: Scenedesmus subspicatus) over several generations.

The study was conducted in accordance with Commission Regulation (EC) No 761/2009 amending Regulation No 440/2008, Method C.3 ‘Freshwater Alga and Cyanobacteria, Growth inhibition test’ (2009) which is equivalent to OECD Guideline for Testing of Chemicals No. 201 (2006).

Exponentially growing algal cells were exposed for a period of 72 hours to a range of concentrations, nominally 4.3, 9.4, 20.7, 45.5, 100 mg/L of Vulkacit P Extra N dissolved in dilution water. Auxiliaries used to prepare the test media were an ultrasonic bath, a magnetic stirrer, a folded filter and an aseptic filter.

The cell densities were measured at 24 hour intervals. Inhibition of the algal population was measured as reduction in growth rate (index r), relative to control cultures grown under identical conditions. Growth rates were also used to calculate a No Observed Effect Concentration (NOEC) and a Lowest Observed Effect Concentration (LOEC) according to Williams Multiple Sequential t-test Procedure. The following values were determined:

Results [mg/L]:

ErC 50*# (0-72 h):       > 4.3

ErC 10*# (0-72 h):       > 4.3    

NOEC [r]* (tα 0.05):       1.7

LOEC [r]* (tα 0.05):       4.3

* Reduction of growth rate (ErCx, NOEC [r]) is the preferred endpoint according to OECD 201 and for regulatory purposes in the EU. Results relating to yield (EyCx, NOEC [y]) were calculated to fulfil regulatory requirements in some countries (but not in the EU) and are given in the results section of this report.

# For Growth rate a statistical evaluation of the EC data is not possible

The content of the test item during the exposure period was verified by Zinc determination.

The results are expressed in terms of geometric mean measured concentrations. The results refer to the test item concentrations calculated on base of the mean measured Zinc values. According to the product information 1 mg/L Zinc equals to 7.01 mg/L test item (molecular weight 458.0 g/mol, empirical formula C18H20N2S4Zn).

Effective concentrations ranged from 4.3 % to 5.0 % of nominal values at 0 hours, and from 1.6 % to 4.0 % of nominal values at 72 hours.

Description of key information

A study was performed to assess the adverse effects of Vulkacit P Extra N on the growth rate (= rate of increase in cell density with time) and the yield (= biomass at time t minus initial biomass) of the planktonic freshwater algal species Desmodesmus subspicatus (former name: Scenedesmus subspicatus) over several generations.

The study was conducted in accordance with Commission Regulation (EC) No 761/2009 amending Regulation No 440/2008, Method C.3 ‘Freshwater Alga and Cyanobacteria, Growth inhibition test’ (2009) which is equivalent to OECD Guideline for Testing of Chemicals No. 201 (2006).

Exponentially growing algal cells were exposed for a period of 72 hours to a range of concentrations, nominally 4.3, 9.4, 20.7, 45.5, 100 mg/L of Vulkacit P Extra N dissolved in dilution water. Auxiliaries used to prepare the test media were an ultrasonic bath, a magnetic stirrer, a folded filter and an aseptic filter.

The cell densities were measured at 24 hour intervals. Inhibition of the algal population was measured as reduction in growth rate (index r), relative to control cultures grown under identical conditions. Growth rates were also used to calculate a No Observed Effect Concentration (NOEC) and a Lowest Observed Effect Concentration (LOEC) according to Williams Multiple Sequential t-test Procedure. The following values were determined:

Results [mg/L]:

ErC 50*# (0-72 h):       > 4.3

ErC 10*# (0-72 h):       > 4.3    

NOEC [r]* (tα 0.05):       1.7

LOEC [r]* (tα 0.05):       4.3

* Reduction of growth rate (ErCx, NOEC [r]) is the preferred endpoint according to OECD 201 and for regulatory purposes in the EU. Results relating to yield (EyCx, NOEC [y]) were calculated to fulfil regulatory requirements in some countries (but not in the EU) and are given in the results section of this report.

# For Growth rate a statistical evaluation of the EC data is not possible

The content of the test item during the exposure period was verified by Zinc determination.

The results are expressed in terms of geometric mean measured concentrations. The results refer to the test item concentrations calculated on base of the mean measured Zinc values. According to the product information 1 mg/L Zinc equals to 7.01 mg/L test item (molecular weight 458.0 g/mol, empirical formula C18H20N2S4Zn).

Effective concentrations ranged from 4.3 % to 5.0 % of nominal values at 0 hours, and from 1.6 % to 4.0 % of nominal values at 72 hours.

Key value for chemical safety assessment

EC50 for freshwater algae:
4.3 mg/L
EC10 or NOEC for freshwater algae:
1.7 mg/L

Additional information

Should read ErC50 > 4.3 mg/L and NOEC[r] = 1.7 mg/L