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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
Study period:
15 October 1993 to 19 October 1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EPA OTS 797.1050 (Algal Toxicity, Tiers I and II)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD 23 Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures
Version / remarks:
Adaptation of tests solutions for poorly soluble substances
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: All test concentrations were sampled
- Sampling method: Samples of the test solutons were evaluated at 0 and 96 hours.
- Sample storage conditions before analysis:
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:Six nominal concentrations were used in the definitive test. The concentrations were, 0.20, 0.40, 0.80, 1.6, 3.2 and 6.4 mg/L prepared as a WAF. Test solutions were pared by weighing appropriate amounts of the test material onto sterile glass coverslips. Each coverslip was placed in a 1 L beaker with a Teflon coated stirrer bar. 1 litre of sterile nutrient medium was added. The solutions were placed on a magnetic stir plate and stirred for 20 hours with a vortex of greater than half of the water column. The control solution was prepared in the same manner as the test solutions. Following stirring, all test solutions were allowed to settle for at least 1 hour. After the test solutions were allowed to settle. The test solutions were siphoned from the lower column using a sterile glass tube and latex tube. Siphoning of the test solution was started at level 1 and proceeded up to level 6. 300 mL of solution were siphoned from each test level. 100 mL at a time, and placed into an appropriately labelled test vessel. After 300 mL of solution were removed, approximately 200 ml were siphoned for water chemistry. 25 mL was using for TOX analysis.
- Controls: Untreated control
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): All solutions at the time of siphoning were clear with no visible surface film.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: Selenastrum capricornutum Printz
- Source (laboratory, culture collection): Department of Botany, Culture Collection of Algae, University of Texas at Austin.
- Age of inoculum (at test initiation): 7 days
- Method of cultivation: The parent stock was grown on an agar slant contained in a 50 mL cuture tube. Cultures were initiated usin a log this parent stock or cloned form an existing culture derived from the parent stock in 100 mL of sterile culture medium.

ACCLIMATION
- Culturing media and conditions (same as test or not): Same as test
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
28 mg/L as CaCO3
Test temperature:
23 °C
pH:
7.3-7.9
Conductivity:
100 Mhos/cm
Nominal and measured concentrations:
Nominal Concentration: Control, 0.20, 0.40, 0.80, 1.6, 3.2 and 6.4 mg/L as WAF
Mean Measured Concentration: Control, 0.19, 0.21, 0.89, 0.93, 1.7 and 4.0 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: Glass (250 mL Erlenmeyer flasks), filled to 100 mL
- Initial cells density: 1E+04 cells/mL in test vessels and 1E+06 cells/mL in control vessels
- No. of vessels per concentration (replicates): Triplicate
- No. of vessels per control (replicates): Triplicate

GROWTH MEDIUM
- Detailed composition:
1 mL of each of the nutrients solutions diluted with 1000 mL of autoclaved reagent water. Reagent water defined as reverse osmosis/deionised water passed through carbon, ion-exchange, and organic absorption cartridges and filtered through a 0.2 µm hollow-fibre final filter to produce 16-18 megohm.cm water.
Macronutrient stock solution:
NaNO3 25.500 g
NaHCO3 15.000 g
MgSO4.7H2O 14.700 g
MgCl2.6H2O 12.164 g
CaCl2.2H2O 4.410 g
K2HPO4 1.044 g
Micronutrients stock solution:
MnCl2.4H2O 415.4 mg
H3BO3 185.5 mg
FeCl3.6H2O 159.8 mg
Na2MoO4.2H2O 7.3 mg
ZnCl2 3.3 mg
CoCl2.6H2O 1.4 mg
CuCl2.2H2O 12.0 µg
Na2EDTA.2H2O 300.0 mg
The medium was re-sterilised by passing through 0.45 µm Millipore filters.
TEST MEDIUM / WATER PARAMETERS
- Total organic carbon: < 1.0 mg/L
- Metals: Aluminium < 0.20 mg/L
Arsenic < 0.010 mg/L
Cadmium < 0.50 µg/L
Chromium < 0.001 mg/L
Cobalt < 0.05 mg/L
Copper < 0.02 mg/L
Iron 0.57 mg/L
Lead < 0.003 mg/L
Mercury < 0.20 mg/L
Nickel < 0.05 mg/L
Silver < 0.50 µg/L
Selenium < 0.005 mg/L
Zinc < 0.04 mg/L
- Alkalinity: 38 mg/L as CaCOs
- Conductivity: 100 Mhos/cm
- Culture medium different from test medium: Cultures were maintained under the same conditions of the toxicity test.
- Intervals of water quality measurement: Biyearly

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: No
- Photoperiod: Continuous
- Light intensity and quality: Cool white fluorescent light. 400 ± 10 % footcandles (approximately 4300 Lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Cell counts were taken at 0, 24, 48, 72 and 96 hours.
- Determination of cell concentrations: counting chamber (haemocytometer)

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 3 range finding tests were conducted
1st Range Finding Test: Nominal test concentrations (prepared as a WAF): 1, 10 and 100 mg/L
2nd Range Finding Test: Nominal test concentrations (prepared as a WAF): 0.01, 0.1 and 1 mg/L
3rd Range Finding Test: Nominal test concentrations (prepared as a WAF): 0.5, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study:
1st Range finding study: Algal cell counts after 96 hours were 0 % of the control population. Definitive test concentrations could not be determined from this test.
2nd Range finding study: Algal cell counts were 116, 134 and 95 % of the control population at 96 hours. A third range finding study was deemed necessary in order to determine definitive test concentrations.
3rd Range finding study: Algal cell counts were 103, 99, 52 and 24 % of the control population at 96 hours. The results of this test were used to determine test concentrations for the definitive test.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
NOELR
Effect conc.:
0.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
2.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95 % CI 1.6 and 3.0 mg/L as WAF
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
2.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95 % CI 1.6 and 2.7 mg/L as WAF
Key result
Duration:
48 h
Dose descriptor:
NOELR
Effect conc.:
1.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
48 h
Dose descriptor:
NOELR
Effect conc.:
0.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
96 h
Dose descriptor:
EL10
Effect conc.:
0.42 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95 % CI 0.16 and 0.69 mg/L as WAF
Key result
Duration:
96 h
Dose descriptor:
EL50
Effect conc.:
1.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95 % CI 1.4 and 2.5 mg/L as WAF
Key result
Duration:
96 h
Dose descriptor:
EL90
Effect conc.:
> 6.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95 % CI 3.0 and >6.4 mg/L as WAF
Key result
Duration:
96 h
Dose descriptor:
NOELR
Effect conc.:
0.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
1.4 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95 % CI 1.0 and 1.7 mg/L as WAF
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.5 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95 % CI 0.98 and 2.1 mg/L as WAF
Key result
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
1.3 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95 % CI 0.88 and 1.8 mg/L as WAF
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.93 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.89 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
0.89 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
96 h
Dose descriptor:
EC10
Effect conc.:
0.29 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95 % CI <0.19 and 0.55 mg/L as WAF
Key result
Duration:
96 h
Dose descriptor:
EC90
Effect conc.:
> 4 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95 % CI <0.19 and >4.0 mg/L as WAF
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
0.89 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The measured test concentrations at 0 hours ranged from 52 % to 130 % of the nominal test concentrations. The mean measured test concentrations for 96 hours ranged from 33 to 140 % of the nominal. The variability between 0 and 96 hour measured concentrations was believed to be related to the viscosity of the test substance and sampling a WAF for a nonwater-soluble test material. Changes in mixing ratios were expected to create non-uniformity in the amounts of the test material in solution. The mean measured test concentrations for this study were 53 to 110 % of the nominal test concentrations.
Recoveries of the quality control fortification samples for the study ranged from 46.4 to 600 % of the nominal fortification samples. Results for the 0 hour low spikes and for the 96 hour low spikes yielded recoveries that were unacceptable high. This was attributed to a weighing error taring the microscope slide.
Low recoveries for the treatment levels and the spikes were believed to be due to the insolubility of the test material in water and all samples settled for a minimum of 1 hour before the WAF was removed.
- Cell growth: Cell growth was demonstrated in each test level at each time point. The test substance was considered to have an algistatic effect on cell growth. The multiple means test (Dunnett’s test) indicated a significant inhibition effect on growth at 0.80, 1.6, 3.2 and 6.4 mg/L WAF (nominal concentration) when compared to the controls at 96 hours.
The ErC50 values could not be determined because the maximum percent inhibition was not greater than 45 % for any time point.
Reported statistics and error estimates:
Residue concentrations:
(µg/mL equiv. from std curve eq.*vol. for analysis mL*dilution factor)/vol. extr. mL=µg/mL
One-way analysis of variance (ANOVA) was conducted with a Dunnett’s comparison to the control. One-tailed Dunnett’s test was conducted at 0.05 significance with an alternate hypothesis of the mean number cells reduced. Levene’s test evaluated homogeneity of variance. If p was greater than 0.01, the cell counts divided by 10⁴. If p was less than 0.01 for some points, the cell count square root was used.
For effect concentration, a logistic model was used with percent inhibition based on growth as the dependent variable and concentration as the independent variable. Percent inhibition was calculated as area under curve (AUC) for and or growth rate.
AUC growth:
A=((N1-N0)/2)*t1+((N1+N2-2N0)/2)*(t2-t2)+((Nn-1+Nn-2N0)/2)*(tn-tn-1)
A=area
Ni=Cell density at the ith time
N0=cell density at 0 hr
ti=time
Percent inhibition:
IA=((Ac-At)/Ac)x100
IA=%inhibition growth
Ac=AUC control
At=AUC test.
Growth rate:
µ=(lnNn–lnN1)/(tn-t1)
µ=av. specific growth rate
Nn=cell density 2nd adjacent time point
N1=cell density 1st adjacent time point
tn=2nd adjacent time point
t1=1st adjacent time point
Percent inhibition:
Iµ =((µc-µt)/µc)*100
Iµ=% inhibition of growth rate
µc= x̄ control growth
µt=growth rate for the test concentration at time
A four-parameter logistic model with two parameters fixed, described dose response.
%inhibition=D+((A-D)/(1+(CONC**(B))*(EC50**(-B)))
A=Min %inhibition at 0%
D=Max %inhibition at 100%
B=slope
Nonlinear modelling estimated EC50 and slope:
EC10=EC50*(((A-10)/(10-D)**(1/B)
EC90=EC50*(((A-90)/(90-D))**(1/B)
X hat method distribution estimated 95% confidence limits. The EC90 95% CL were disregarded as the values didn’t approach that level. Two goodness of fit measures were calculated. The R² for the percent of variation about the mean using the logistic model. The RMSE for the average distance between the observed data values and model fit.

Table 1: Cell counts

Nominal Test Concentration

(mg/L as WAF)

Mean measured

Concentration (mg/L)

Mean Cell Counts 10⁴ cells/mL

0 hours

24 hours

48 hours

72 hours

96 hours

Control

Control

1.0

2.2

6.5

28

110.0

0.2

0.19

2.0

6.0

24

97

0.4

0.21

1.7*

6.4

29

110

0.8

0.89

1.7*

7.5

24

98

1.6

0.93

1.4*

4.9

12*

42*

3.2

1.7

0.93*

3.4*

12*

33*

6.4

4.0

0.85*

2.6*

7.7*

27*

* statistically significant inhibition (p≤0.05)

 

Table 2: Nominal and measured concentrations of the test solutions

Nominal Concentration

(mg/L as WAF)

0 Hours

96 Hours

% Loss*

Mean Measured Concentration

(mg/L)

% Nominal

Measured Concentration

(mg/L as WAF)

% Nominal

Measured Concentration

(mg/L as WAF)

% Nominal

Control

<0.0298

-

0.0403

-

-

N/A

N/A

0.2

0.264

130

0.119

60

70

0.19

95

0.4

0.292

73

0.13

33

40

0.21

53

0.8

0.64

80

1.14

140

#

0.89

110

1.6

1.2

75

0.653

41

34

0.93

58

3.2

1.99

62

1.32

41

21

1.7

53

6.4

3.32

52

4.75

74

#

4

63

Results for 96 hours are from rederivatised samples, which confirmed the results from the original derivatised samples.

*0 hour nominal - 96 hour nominal. Variability in loss of measured concentration between hour 0 and hour 96 is believed to be related to sampling a water-accommodated fraction for a nonwater-soluble test material.
# A gain was observed which may be attributed to the viscosity of the test substance and the method for sampling a water accommodated fraction for a nonwater-soluble test substance.

 

Table 3: Fortifications and recoveries of the test substance in quality control samples

Sample

0 Hour

96 Hours

Nominal Concentration

(mg/L as WAF)

Measured Concentration

(mg/L)

% Nominal

Nominal Concentration

(mg/L as WAF)

Measured Concentration

(mg/L)

% Nominal

Low Spike A*

0.1

0.625

600

0.1

0.135

100

Low Spike B*

0.1

0.536

500

N/A

N/A

N/A

Mid Spike A

1

0.612

61

1

1.01

100

Mid Spike B

1

0.666

67

N/A

N/A

N/A

High Spike A

10

5.07

50.7

10

6.55

65.5

High Spike B

10

4.64

46.4

N/A

N/A

N/A

The number of significant figures in the nominal concentrations of the spike samples varied due to the precision of the balance used to weigh the test material to prepare the spike samples.
* Results were unacceptable high because the precision of the balance was to four decimal places and 0.0001 g of test material was weighed to prepare the low spike samples.

Validity criteria fulfilled:
yes
Conclusions:
The EbC50 (96 hours) for the test material based on nominal test concentrations was calcualted to be 1.9 mg/L as WAF and based on mean measured concentrations, 1.3 mg/L. After 96 hours, the NOEC was 0.80 mg/L as WAF (nominal test concentration) and 0.89 mg/L (mean measured concentration) based on the absence of a growth inhibition effect.
Executive summary:

The toxicity of the test material to algae was evaluated using a test conducted in accordance with EPA OTS 797.1050 and OECD 201.

Cell growth was demonstrated in each test level at each time point. The test substance was considered to have an algistatic effect on cell growth. The multiple means test (Dunnett’s test) indicated a significant inhibition effect on growth at 0.80, 1.6, 3.2 and 6.4 mg/L WAF (nominal concentration) when compared to the controls at 96 hours.

The ErC50 values could not be determined because the maximum percent inhibition was not greater than 45 % for any time point.

The EbC50 (96 hours) for the test material based on nominal test concentrations was calculated to be 1.9 mg/L as WAF and based on mean measured concentrations, 1.3 mg/L. After 96 hours, the NOEC was 0.80 mg/L as WAF (nominal test concentration) and 0.89 mg/L (mean measured concentration) based on the absence of a growth inhibition effect.

Description of key information

The toxicity of the test material to algae was evaluated using a test conducted in accordance with EPA OTS 797.1050 and OECD 201.

Cell growth was demonstrated in each test level at each time point. The test substance was considered to have an algistatic effect on cell growth. The multiple means test (Dunnett’s test) indicated a significant inhibition effect on growth at 0.80, 1.6, 3.2 and 6.4 mg/L WAF (nominal concentration) when compared to the controls at 96 hours.

The ErC50 values could not be determined because the maximum percent inhibition was not greater than 45 % for any time point.

The EbC50 (96 hours) for the test material based on nominal test concentrations was calculated to be 1.9 mg/L as WAF and based on mean measured concentrations, 1.3 mg/L. After 96 hours, the NOEC was 0.80 mg/L as WAF (nominal test concentration) and 0.89 mg/L (mean measured concentration) based on the absence of a growth inhibition effect.

Key value for chemical safety assessment

EC50 for freshwater algae:
1.9 mg/L
EC10 or NOEC for freshwater algae:
0.89 mg/L

Additional information