mecoprop-P (ISO); (R)-2-(4-chloro-2-methylphenoxy)propionic acid

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:

November 1992 to March 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

14th March 1990

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The mean concentrations of the test material in the water samples from November 23 and 26, 1992 were found to be in the range of 97.4 - 105.2 % of the nominal concentrations for the 3, 81 and 729 mg/L samples, respectively.
- Sample storage conditions before analysis: Refrigerator
- Sampling method: Spot checks were taken for concentration control analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The stock solution was prepared by adding 1.6269 g of the test material to 500 mL algal nutrient solution.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: SAG B 61.81
- Method of cultivation: The test material was added to algal medium (final volume of 60 mL) in 100 mL Erlenmeyer dimple flasks which was inoculated with algae from a pre-culture to get an initial cell concentration of about 3 x E^04 cells/mL.

ACCLIMATION
- Culturing media and conditions: Algal medium.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No

Test temperature:

23 ± 1 ºC

pH:

7.9

Nominal and measured concentrations:

Nominal concentrations: 3, 9, 27, 81, 243, and 729 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer
- Material, size, headspace, fill volume: 100 mL Erlenmeyer dimple glass flasks, final fill volume 60 mL.
- No. of organisms per vessel: 3 x E^04 cells/mL.
- No. of vessels per concentration: For each test concentration five replicates were carried out.
- No. of vessels per control: 10 replicates
- Other: The culture flasks were placed in a temperature controlled incubator until sampling. During the experiment the algae were kept in suspension by constant shaking at 125 rpm.

GROWTH MEDIUM
- Standard medium used: No
- Detailed composition if non-standard medium was used:
Stock solutions, all constituents dissolved in water
Solution No. 1: NH4Cl, 1.500 g/10 mL
Solution No. 2: MgCl2·6H20,1.200 g/10 mL
Solution No. 3: CaCl2 ·2H20 1.800 g/10 mL
Solution No. 4: MgS04·7H20 1.500 g/10 mL
Solution No. 5: KH 2PO4, 0.160 g/10 mL
Solution No. 6: FeCl3·6H20, 0.160 g/100 mL
Solution No. 7: Na 2EDTA·2H2O, 0.200 g/100 mL
Solution No. 8: H3BO3, 0.370 g/100 mL
Solution No. 9: MnCl2·4H20, 0.415 g/100 mL
Solution No. 10: ZnCl2, 0.600 g/100 mL
Solution No. 11: CoCl2·6H2O, 0.300 g/100 mL
Solution No. 12: CuCl2·2H2O, 0.100 g/10 mL
Solution No. 13: Na2MoO4·2H20, 0.140 g/10 mL
Solution No. 14: NaHC03, 0.500 g/10 mL

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
The following solutions were added to 950 mL of destilled water and finally adjusted up to 1 000 mL with distilled water:
Solution No. 1 : 0.100 mL
Solution No. 2: 0.100 mL
Solution No. 3: 0.100 mL
Solution No. 4: 0.100 mL
Solution No. 5: 0.100 mL
Solution No. 6: 0.050 mL
Solution No. 7: 0.050 mL
Solution No. 8: 0.050 mL
Solution No. 9: 0. 00 mL
Solution No. 10: 0.050 mL (prior diluted 1:100)
Solution No. 11: 0.050 mL (prior diluted 1:100)
Solution No. 12: 0.010 mL (prior diluted 1:10^4 )
Solution No. 13: 0.050 mL (prior diluted 1:100)
Solution No. 14: 1.000 ml
The solution was filter-sterilised using a "Sartorius Membranfilter" system SM 16268 with a triple layer of membran-filters with decreasing pore size from 0.6, 0.45, to 0.2 μm, each separated from the other by one layer of cheesecloth.

OTHER TEST CONDITIONS
- Sterile test conditions: Yes. the sterility of the nutrient solution was checked by plating aliquots on agar plates and incubating the plates for 48 h at 28 ºC. Only nutrient solutions giving negative results (no detectable germs after the incubation period) were used.
- Adjustment of pH: After preparing the nutrient solution, the pH was determined to be 7.9
- Photoperiod: Continuous uniform illumination was obtained by universal whitetype fluorescent lamps.
- Light intensity and quality: 8 000 lux

EFFECT PARAMETERS MEASURED : The effect of the test material on the growth of the green alga Pseudokirchneriella subcapitata was investigated.
- Determination of cell concentrations: The cell concentration in each flask was determined 24, 48, and 72 hours after starting the experiment by using a photometer (623 nm, 2 cm glass cuvettes). For the control algal medium was used as a blank. For each concentration additional blanks were used containing the corresponding test material concentrations. To obtain the actual number of cells/mL a calibration curve of cell numbers (counted under a microscope) versus extinction values was established.
To obtain the actual number of cells/L a calibration curve of cell numbers (counted under a microscope) versus extinction values was established.
At each sampling interval one aliquot of each of the replicates of test and control flasks was collected for evaluation of algal densities. Subsequently the mean values per treatment were calculated and growth curves plotted.
- At the last sampling interval, the pH of all individual samples (controls as well as treated samples) were measured.

TEST CONCENTRATIONS
- Test concentrations: For the test the following concentrations (nominal) were selected: 3, 9, 27, 81, 243, and 729 mg/L.
- Range finding study: The concentration range over which effects are likely to occur was determined on the basis of a range finding test (prior to the definitive study not according to GLP).

CALCULATIONS AND DETERMINATION OF EC50
At each sampling interval one aliquot of each of the replicates of test and control flasks was collected for evaluation of algal densities. Subsequently the mean values per treatment were calculated and growth curves plotted.
In order to determine the concentration/effect-relationship, the area below the growth curves was calculated using the following formula:

A = ((N1 – N0) / 2) x t1 + ((N1 + N2 – 2N0) / 2) x (t2 – t1) + ((Nn-1 + Nn – 2N0) / 2) x (tn – tn-1)

where
A = Area
N0 = Number of cells /mL at t0.
Nl = Measured number of cells/mL at t1.
Nn = Measured number of cells/mL at tn.
t1 = Time of first measurement after beginning of test.
tn = Time of nth measurement after beginning of test.
The percent inhibition of the cell growth at each test concentration (IA) was calculated from the difference between the area under the control growth curve (Ac) and the area under the growth curve at each test concentration (AT):

IA (%) = ((AC – AT) / AC) x 100

In addition the growth rate is determined according to the formula:

μ1 = 1nNt – 1nNt-1 for the individual daily growth rate, and
μ = 1nNt – 1nNo / t for the overall average daily growth rate

Where:
t = Number of days

The percent inhibition of the growth rate at each test concentration (IR) is calculated from the difference between the average control growth rate (μc) and the average growth rates at each test concentration (μT):

IR (%) = ((μC – μT) / μC) x 100

The % inhibition results from growth curves IA or growth rates IR are used to determine the EC50 (0 - 72 hours) by plotting the inhibition values against the corresponding concentrations and connecting the points by a straight line or using a regression curve (PC with software "Harvard Graphics"). The EC50 value results from the intercept of the curve with the parallel to the abscissa at 50 % inhibition (graphical interpolation). The EC10 can be determined accordingly at the 10 % inhibition line.
The NOEC can be determined statistically using a Dunnett's test. The calculations for this are carried out with a PC and the software package "RS/1".

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 729 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

270 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

145 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

35 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

Based on nominal concentrations (measured values are > 80 % of nominal ones) the following results with respect to algal biomass 'b' and average growth rate 'r' are derived graphically.
ECb50 (0-72 h) = 270 mg/L
ECb10 (0-72 h) = 35 mg/L
ECr50 (0-72 h) > 729 mg/L
ECr10 (0-72 h) = 145 mg/L
The NOECb (95 % probability) was determined with a Dunnett's test to be 27 mg/L.
At 243 mg/L about 50 % and at 729 mg/L 100 % of the algae showed morphological changes (the algae were bigger than those in the control).

Reported statistics and error estimates:

The % inhibition results from growth curves IA or growth rates IR are used to determine the EC50 (0 - 72 hours) by plotting the inhibition values against the corresponding concentrations and connecting the points by a straight line or using a regression curve (PC with software "Harvard Graphics"). The EC50 value results from the intercept of the curve with the parallel to the abscissa at 50 % inhibition (graphical interpolation). The EC10 can be determined accordingly at the 10 % inhibition line.
The NOEC can be determined statistically using a Dunnett's test. The calculations for this are carried out with a PC and the software package "RS/1"

Effect of the Test Material on the Growth of Pseudokirchneriella subcapitata.

Concentration of Test Material (mg/L)	% Inhibition in 72 h (Biomass)	% Inhibition in 72 h (Growth Rate)
3	-6.5	-1.6
9	-5.0	-0.9
27	7.9	1.7
81	18.2	5.0
243	46.7	14.5
729	80.6	35.4

Negative values indicate stimulated growth.

Validity criteria fulfilled:

not specified

Conclusions:

Under the conditions of the study the 72 h ECr50 (growth rate) was > 729 mg/L.  The 72 h ECr10 was 145 mg/L. The equivalent rates based on biomass were 270 mg/L (ECb50 ) and 35 mg/L (ECb10).  The NOECb (95 % probability) was 27 mg/L.

Executive summary:

The acute toxicity of the test material on the growth of the green alga Pseudokirchneriella subcapitata was assessed according to OECD Test Guidline 201 and in compliance with GLP.

Under the conditions of the study the 72 h ECr50 (growth rate) was > 729 mg/L.  The 72 h ECr10 was 145 mg/L. The equivalent rates based on biomass were 270 mg/L (ECb50 ) and 35 mg/L (ECb10).  The NOECb (95 % probability) was 27 mg/L.

Description of key information

Dohmen (1993)

Under the conditions of the study the 72 h ECr50 (growth rate) was > 729 mg/L.  The 72 h ECr10 was 145 mg/L. The equivalent rates based on biomass were 270 mg/L (ECb50 ) and 35 mg/L (ECb10).  The NOECb (95 % probability) was 27 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

729 mg/L

EC10 or NOEC for freshwater algae:

145 mg/L

Additional information

Dohmen (1993)

The acute toxicity of the test material on the growth of the green algaPseudokirchneriella subcapitatawas assessed according to OECD Test Guidline 201 and in compliance with GLP. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Under the conditions of the study the 72 h ECr50 (growth rate) was > 729 mg/L.  The 72 h ECr10 was 145 mg/L. The equivalent rates based on biomass were 270 mg/L (ECb50 ) and 35 mg/L (ECb10).  The NOECb (95 % probability) was 27 mg/L.