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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April - September 2014
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:
adopted 23 March 2006
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 12.0, 27.8, 53.4, 97.4, and 180 µg ziram/L
- Sampling method: Samples at 24 and 48 hours were taken from single, sacrificial replicates designated for analytical measurements only. At exposure termination, samples were collected from the pooled replicates from each treatment and control group.
- Sample storage conditions before analysis: At each interval, samples were collected directly into amber glass vials and processed immediately for analysis. Two sets of samples were collected at each interval. One set was immediately analyzed and the second set was stabilized and stored frozen for possible future analysis.
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 3000 µg ziram/mL in DMF was prepared. Each test solution was prepared by diluting 100 µL of each respective stock in 1000 mL of freshwater AAP medium.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): N,N-dimethylformamide (DMF)
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): The stock was serially diluted with DMF to prepare four additional stocks at target nominal concentrations of 188, 375, 750 and 1500 µg ziram/mL. Each test solution was prepared by diluting 100 µL of each respective stock in 1000 mL of freshwater AAP medium.
- Controls: The negative control solution consisted of freshwater AAP medium without test substance added. The solvent control solution contained 0.1 mL DMF/L, which was equivalent to the solvent concentration in all of the treatment groups.
- Evidence of undissolved material (e.g. precipitate, surface film, etc):
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain:
- Source (laboratory, culture collection): Original algal cultures were obtained from the University of Toronto, and have been maintained in culture medium at Wildlife International, Easton, Maryland since March 2000
- Age of inoculum (at test initiation): Algal cells for this study were taken from a culture that had been transferred to fresh medium four days prior to test
initiation
- Method of cultivation: The algal cells were cultured and tested in freshwater AAP medium. The pH of the medium was adjusted to 7.5. Test chambers were held in an environmental chamber at a temperature of 24 ± 2ºC. The target light intensity was 6000 lux ± 10%.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
none
Hardness:
Freshwater AAP medium
Test temperature:
24 ± 2ºC
pH:
7.5
Dissolved oxygen:
not reported
Salinity:
not reported
Nominal and measured concentrations:
Nominal: 18.8, 37.5, 75.0, 150, 300 µg/L
Measured: 12.0, 27.8, 53.4, 97.4, 180 µg/L (geomean)
Details on test conditions:
TEST SYSTEM
- Test vessel: sterile, 250-mL glass Erlenmeyer flasks plugged with sterile foam stoppers
- Type (delete if not applicable): closed
- Fill volume: 100 mL
- Aeration: mechanical shaker at 100 rpm
- Initial cells density: 10,000 cells/mL
- Control end cells density: Negative Control: 1,521,867 cells/mL; Solvent Control 1,645,964 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: purified well water
- Metals: checked
- Pesticides: checked
- Chlorine: checked
- Ca/Mg ratio: 2.62
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: adjusted to 7.5
- Photoperiod: constant illumination
- Light intensity and quality:
- Salinity (for marine algae):

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations:electronic particle counter
- Chlorophyll measurement: no

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study
- Test concentrations: 10, 30, 100, 300 µg/L
- Results used to determine the conditions for the definitive study: 34% and 99% inhibition at 100 and 300 µg/L, respectively
Reference substance (positive control):
yes
Remarks:
A test with a reference substance is performed at least once a year.
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
53.4 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
94 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI: 90.0 to 97.7 µg/L
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
74.4 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 95% Cl: 69.8 to 79.2 µg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
65.8 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI: 60.9 to 71.2 µg/L
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
not reported
Reported statistics and error estimates:
see 95% CI given in results table

Validity criteria:

Mean cell density in the control flasks increased by a factor of 206 after three days, achieving the 16x growth criterion.

The coefficient of variation of average specific growth rates in the control replicates was 2.7%, achieving the less than 7% criterion.

The mean percent coefficient of variation forsection-by-section specific growth rates in the control replicates was 21.3%, achieving the less than 35% criterion

Validity criteria fulfilled:
yes
Conclusions:
The 72-hour EC50 values for growth rate and yield were determined to be 73.3 µg a.s./L and 94.0 µg a.s./L, respectively. The 72 hour NOEC values for growth rate and yield were both determined to be 53.4 µg a.s./L.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2 June 1995- 24 October 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study; results based on biomass
Qualifier:
according to guideline
Guideline:
other: FIFRA Guideline Number: 122-2
Qualifier:
according to guideline
Guideline:
other: FIFRA Guideline Number: 123-2
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- At test initiation and test termination (day 5) a single sample was collected from each control and test solutions and analyzed for Ziram concentration, based on measurement of 14C residues. Three Quality Control (TQC) samples were prepared at test initiation and termination using fresh algal growth medium. In addition to the exposure solution analyses, triplicate samplesof the Ziram stock solution at test initiation for confirmation of parent Ziram concentration.
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A primary stock solution of 14C Ziram was first prepared by dissolving the entire amount of 14C Ziram received (5 mCi) in dimethylformamide, to a total volume of 50 mL. Triplicate 10 uL aliquots of this primary stock solution were assayed by liquid scintillation counting (LSC) which yielded a mean concentration of 0.569 mg a.i/mL. Furthermore, a 6 mg a.i/mL stock solution (combination of radiolabelled and non radiolabelled Ziram) was prepared by dissolving 0.304g of non radiolabelled Ziram (0.2979 g as a.i) and 0.35 mL of the 0.569 mg a.i/mL radio labelled stock solution (1.992 mg) with DMF to a volume of 50 mL. Then, a 50 uL aliquot of the 6 mg a.i/mL stock solution was further diluted to 500 mL with esterile AAP medium, to prepare the highest nominal concentration. Appropriate volumes of this solution were diluted to 10 mL with DMF to prepare secondary stock solutions (for the other nominal concentrations). Aliquots of 50 uL of each secondary stock solution were the diluted to 500 mL to give the final nominal concentrations to be tested.
- Controls: control and solvent control
- Chemical name of vehicle: dimethylformamide (DMF)
- Concentration of vehicle in test medium: 0.10 uL/mL
- Evidence of undissolved material: the solutions appeared to be clear and colourless without any signs of undissolved material
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: 1648
- Source: Carolina Biological Supply Company, Burlington, North Carolina, USA

ACCLIMATION
- Culturing media and conditions: the culture medium used was Algal Assay Procedure (AAP) medium prepared with esterile deionized water. The pH of this culture medium was adjusted to 7.5 +/-1 and stock cultures were grown in 125 mL flasks containing 50 mL medium. The stock cultures were maintained within the following conditions for a minimum of 3 days prior to test initiation: a shaking rate of 100 +/- 10 rpm, 24 +/- 1°C temperature and continuous illumination at the surface of the medium at a intensity of 3200 to 5000 lux.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
5 d
Test temperature:
24 +/- 1°C
pH:
7.5-8.8
Nominal and measured concentrations:
Nominal concentrations: 0.039, 0.075, 0.15, 0.30, and 0.60 mg a.i/L
Measured concentrations: 0.038, 0.072, 0.14, 0.27 and 0.48 mg a.i/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 125 mL Erlenmayer flasks, containing 50 mL test medium
- Initial cells density: 0.3 x104 cells/mL
- Control end cells density: 270x104 and 331x104 (control and solvent control)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- No. of vessels per vehicle control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes, AAP medium

OTHER TEST CONDITIONS
- Photoperiod: continuous illumination
- Light intensity: 3200-5400 lux
- Conductivity: 90-110 umhos/cm

EFFECT PARAMETERS MEASURED :
- Determination of cell desity at days 1, 2, 3, 4 and 5.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: 5 to 10 April 1995
- Test concentrations: 0.0060, 0.060, 0.6 and 6 mg/L
- Results used to determine the conditions for the definitive study: cell densities averaged 138, 125, 0.13 and 0x104 cells/mL respectively (low to
high concentration)
Duration:
5 d
Dose descriptor:
EC50
Effect conc.:
0.066 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
5 d
Dose descriptor:
NOEC
Effect conc.:
0.038 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Reported statistics and error estimates:
William's test, Shapiro-Wilks normality test, Bettler's homogeneity of variance test (95% or 99% level of certainty)
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
4 May 2006- 3 July 2007
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: Relevant methodological deficiencies
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
actual test substance concentrations were estimated but not measured
GLP compliance:
yes (incl. QA statement)
Remarks:
The Swiss GLP Monitoring Authorities
Analytical monitoring:
yes
Details on sampling:
- The concentration of active ingredient was measured at test start and test end in order to provide an evidence that the concentration of the test substance being tested was satisfactorily maintained throughout. As the test was carried out at low concentrations, analytical measurements of active ingredient were not possible in the actual exposure concentrations. Therefore measurement of concentration of active and extrapolation of the results were performed as described in ECETOC (1996). Analysis of active ingredient was performed only in the stock solution (22.40 mg/L) which was exposed without test organisms at the same conditions as actual test solutions. Stock solution was measured at 0 and 72 hours and the results were extrapolated to the concentrations used in the definitive test.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test substance and the stock solution were dissolved with sterile deionized water and the test solutions with sterile liquid medium (L.C. Oligo). Test solutions were prepared only at test start. A portion of each test solution (10 mL) was mixed with sterile liquid medium (89 mL) and algae suspension (1.0 mL) and distributed per flask (total volume per flask was 100 mL)
- Controls: sterile liquid medium (L.C. Oligo), sterile deionized water and algae inoculum
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Green algae
- Source: CETESB (Sao Paulo, Brazil)

ACCLIMATION
- Acclimation period/ Culturing media and conditions: Algae from stock cultures in solid medium were transferred to a 100 mL sterile liquid medium (L. C. Oligo) and were kept in the growth chamber for 3 days in the same environmental conditions as in the test. Liquid culture was centrifuged in glass tubes on the first day of testing for 15 minutes at 1500 rpm. Sediment was suspended in 15 mL of sterile liquid medium (L. C. Oligo). This procedure was repeated twice and cell density was determined in a spectrophotometer HACH DR/2000
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
24.5 +/- 0.5°C
pH:
Control: 7.39- 9.40
Treatment groups: 6.52- 8.16
Nominal and measured concentrations:
Nominal concentrations: 0.14mg/L, 0.28 mg/L, 0.56 mg/L, 1.12mg/L and 2.24 mg/L
Nominal concentrations active ingredient: 1.70 mg/L, 0.85 mg/L, 0.43 mg/L, 0.21 mg/L, 0.11 mg a.i./L
Extrapolated concentrations from stock solution measurements, active ingredient (initial-final): 1.74-1.75 mg/L, 0.87-0.88 mg/L, 0.43-0.44 mg/L, 0.22-0.22 mg/L, 0.11-0.11 mg a.i./L
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL Erlenmayer flasks
- Initial cells density: ca. 2 x 10exp4 cells/mL
- Control end cells density: 16.2 x 10exp5
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Detailed composition medium: L.C. Oligo medium:

Ca(N03)2.4H20 40.0 mg/L
KNO3 100.0 mg/L
MgSO4.7H20 30.0 mg/L
K2HPO4 40.0 mg/L
CuSO4.5H20 0.015 mg/L
(NH4)6Mo7O24.4H20 0.030 mg/L
ZnSO4.7H20 0.030 mg/L
CoC12.6H20 0.030 mg/L
Mn(N03)2.4H20 0.030 mg/L
C6H802.H20 0.030 mg/L
H3B03 0.030
C6H5FeO7.5H20 0.8 125
FeC13.6H20 0.3 125
FeSO4.7H20 0.3 125
NaHCO3 15.0


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: sterile deionized water

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: 3490-5820 lux, fluorescent light


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study
- Test concentrations: 0.001 mg/L; 0.010mg/L; 0.100 mg/L and 1.00mg/L
- Results used to determine the conditions for the definitive study: Range finding test showed inhibition of growth rate higher than 50% at 1 .00mg/L and 0.010 mg/L after 72 h
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.28 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.56 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.5 mg/L
Nominal / measured:
estimated
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Results with reference substance (positive control):
The 96-h EC50 using data from several accumulated tests was 3.79 mg K2Cr2O7/L, with limits (mean ± 2.SD) of 1.31 and 6.27mg K2Cr2O7/L
Reported statistics and error estimates:
Chi-square test for normality (USEPA,2002)
Bartlett's test for homogeneity of variance (USEPA, 2002)
William's test (Gerbert et al., 1985; Williams, 1971, 1972)

Validity criteria fulfilled:

1. Control cultures increased by a factor higher than 16 within 72 hours

2. Coefficient of variation of average specific growth rates in the control was 7%

3. The mean coefficient of variation of the specific growth rates section-by-section in the control was 13%

Validity criteria fulfilled:
yes

Description of key information

NOErC (72h) = 0.0534 mg a.i./L (Pseudokirchnerella subcapitata )

ErC50 (72 h) = 0.0733 mg a.i./L (Pseudokirchnerella subcapitata)

Key value for chemical safety assessment

EC50 for freshwater algae:
0.073 mg/L
EC10 or NOEC for freshwater algae:
0.053 mg/L

Additional information

The toxicity of zinc bis dimethyldithiocarbamate (CAS No. 137-30-4) to algae species was investigated in three studies. The test chosen as key study (Dobbins et al., 2014) was conducted according to OECD Guideline No. 201, Algae, Growth Inhibition Test (2006), under GLP conditions. Pseudokirchnerella subcapitata was exposed for 72 hours to the test substance and effects on biomass and growth were measured. The resulting ErC50 and NOErC values based on growth inhibition were determined to be 0.0733 and 0.0534 mg a.i./L respectively.

Another study on Pseudokirchnerella subcapitata (Lopes Morandi, 2007) was also conducted according to OECD 201, but the validity of the test was impaired by unreliable analysis of the test material in medium.

An additional study conducted according to FIFRA Guidelines 122-2 and 123-2 (1996) for 120 hours is available (Hoberg, 1995). The results from this test are expressed on the basis of algae biomass, resulting in an EbC50 = 0.066 mg a.i./L.