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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:
Study was carried out between 6 and 9 July 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study according OECD Guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of the signature 08.10.2010
Analytical monitoring:
yes
Details on sampling:
- Test water was sampled and pH and salinity were measured by the study laboratory.
- Sample storage conditions before analysis: Corresponding to the storage condition of test material.
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test solutions were prepared by the addition of a known quantity of mixed ash directly to 1 litre volumes of culture medium. These solutions were stirred for overnight using magnetic followers and then left to stand for 15 minutes. After this time the solutions were filtered through 0.45 µm cellulose nitrate filters, which had been previously washed with 100 mL Reverse Osmosis (RO) water.
- Controls: The control consisted of culture medium only.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): None

The appropriate test solution (100 mL volume) was dispensed to each test and blank vessel.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: unicellular green alga Pseudokirchneriella subcapitata
- Strain: ATCC 22662
- Source (laboratory, culture collection): Brixham Environmental Laboratory culture from the strain
- Age of inoculum (at test initiation): A four day old culture of the alga in the exponential growth phase
- Method of cultivation: The culture was grown in the medium, and under the environmental conditions, described for the test.

ACCLIMATION
- Culturing media and conditions (same as test or not): The culture medium used for the test, and for the maintenance of cultures of the alga used as inoculum for the test was AAP-medium, the compounds of which are shown in the table 1.
Test type:
static
Water media type:
freshwater
Total exposure duration:
72 h
Hardness:
NA
Test temperature:
22 ± 2°C.
pH:
7.6 - 7.7.
Dissolved oxygen:
NA
Salinity:
0 - 0.1‰.
Nominal and measured concentrations:
3.2, 5.6, 10, 18, 32 and 56 mg L-1
Details on test conditions:
TEST SYSTEM
- Test vessel: conical flasks closed with foam bungs
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: glass, 250 ml, 100 mL of test solution
- Aeration: No
- Shaking: orbital shaking 160 rpm
- Initial cells density: 0.547 × 104 cells mL-1 (nominal cell density of 0.5 × 104 cells mL-1).
- No. of vessels per concentration (replicates): triplicate cultures of each concentration of mixed ash
- No. of vessels per control (replicates): Six replicate cultures of the culture medium control
- No. of vessels per vehicle control (replicates): One blank vessel (without algal inoculum) was incubated concurrently for each test concentration and the control.
- Blanks: One

GROWTH MEDIUM
- Standard medium used: AAP medium
- Detailed composition if non-standard medium was used: See Table 1.

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: No
- Intervals of water quality measurement: pH was measured at the start and at the end of the test from all vessels. Salinity was measuren corespondingly, but from three test concentrations ( 3.2, 18 and 56 mg L-1). The temperature of the incubator was continuously monitored using an electronic recording system with hourly recording of values. The light intensity was measured once during the study, in each of four representative positions, using a photometer reading in lux (cosine).

OTHER TEST CONDITIONS
- Adjustment of pH: Adjusted from 7.4 - 10.2 with 1 M hydrochloric acid to 7.4 - 7.5.
- Light intensity and quality: Continuous "cool-white" illumination of 6440 lux ± 13%

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: electronic particle counter Coulter counter.
- Observation intervals: 24, 48 and 72 hours.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Test concentrations: nominal mixed ash concentrations of 3.2, 5.6, 10, 18, 32 and 56 mg L-1
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
5.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
Yield
Remarks on result:
other: p = 0.05
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
Yield
Remarks on result:
other: p = 0.05
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
8.53 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
Yield
Remarks on result:
other: 95% CL 8.10-8.77 mg/L
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
6.52 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
Yield
Remarks on result:
other: 95% CL 5.74-6.92 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
5.85 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
Yield
Remarks on result:
other: 95% CL 1.10-6.33 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
5.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: p = 0.05
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: p = 0.05
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
21.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL 21.1-21.4 mg/L
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
8.78 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL 8.34-9.21 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
7.06 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL 6.62-7.33 mg/L
Details on results:
- Exponential growth in the control (for algal test): Yes
- Observation of abnormalities (for algal test): At 32 mg L-1 some of the cells were observed to be enlarged, clumping and with ruptured cell walls. At 56 mg L-1 clumping of the cells was observed.
- Any stimulation of growth found in any treatment: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None

Biological data

Algal cell particle densities (cell particles per unit volume) were measured as a surrogate for biomass. The algal cell particle densities measured at each time period are given in Table 2. The means of these values are also shown in Table 2 and are plotted as growth curves in Figure 1.

Table 2 Algal cell densities

 

 Nominal loading concentration of mixed ash (mg L-1)  Replicate       Algal cell particle density (104 cells mL-1
     24 hours  48 hours  72 hours
 Culture Medium  A  2.220  10.50 53.20
   B  2.320  12.60 57.90
   C  2.160  11.70 52.20
   D  2.410  12.60 54.80
   E  2.490 13.10 61.40
   F  2.290  12.00  57.40
   Mean  2.315  12.08  56.15
 3.2  A  2.260  10.40  42.80
   B  2.290  12.50  58.90
   C  2.500  11.70  55.20
   Mean  2.350  11.53  52.30
 5.6  A  2.380  11.80  50.20
   B  2.150  12.50  60.00
   C  1.810  11.40  48.80
   Mean  2.113  11.90  53.00
 10  A  0.1980  2.080  14.10
   B  0.1840  2.480  18.00
   C  0.2700  2.610  16.20
   Mean  0.2173  2.390  16.10
 18  A  0.2620  2.180  11.30
   B  0.1100  2.570  10.90
   C  0.08400  2.630  11.50
   Mean  0.1520  2.460  11.23
 32  A  0.2240  0.00001  0.00001
   B  0.1800  0.00001  0.00001
   C  0.2240  0.00001  0.00001
   Mean  0.2093  0.00001  0.00001
 56  A  0.1260  0.00001  0.00001
   B  0.00001  0.00001  0.00001
   C  0.00001  0.00001  0.00001
   Mean  0.04201  0.00001  0.00001

Yield This response is defined as the biomass at the end of the test minus the starting biomass. For the purposes of calculation, the cell particle density count (cell particles per unit volume) is an acceptable surrogate for biomass.These cell particle densities were examined by one-way analysis of variance and Dunnett’s procedure was used to identify significant differences (p = 0.05) from the control. The test substance concentrations yields are given in Table 3, together with the yields expressed as percentages of thecontrol and significant differences from the control. A Weibull model was fitted to the percent effect and log concentration data. This model was then used to estimate the median effective concentration (based on yield curve area, EyC50) and its 95% confidence limits.

Table 3 Mean yields over the test period

  Nominal loading concentration of mixed ash (mg L-1) Mean yield (0 -72 hours)  Percentage of control
 Control  55.60  -
 3.2  51.75  93
 5.6  52.45  94
 10  15.55*  28
 18  10.69*  19
 32  -0.547*  -1
 56  -0.547*  -1

* Significant difference (P=0.05) from the control

Growth rates

The growth rate (0 to72hours) was calculated for each replicate culture, according to the formula.

Growth rate = (Logn(N2/N1))/t

where

N1 = Cell density at start of test

N2 = Celldensity at end of test

t = Time interval (days)

These data were analysed as described for the yield method. The mean growth rates,are given in Table 4, together with the rates expressed as percentages of the control.

A Weibull model was fitted to the percent effect and log concentration data. This model was then used to estimate the median effective concentration (based on yield curve area, EyC50) and its 95% confidence limits.

Table 4 Mean growth rates over the test period

 Nominal loading concentration of mixed ash (mg L-1) Mean daily growth (0 -72 hours)  Percentage of control
 Control  1.544  -
 3.2  1.520  98
 5.6  1.525  99
 10  1.127*  73
 18  1.007*  65
 32  -3.637*  -235
 56  -3.637*  -235

* Significant difference (P=0.05) from the control

Physical and chemical data At the start of the test the pH ranged from 7.4 to 10.2, before adjustment, and 7.4 to 7.6, after adjustment, and at the end of the test the range was from 7.6 to 7.7 (Table 5). No pH shifts were observed in any sample. The hourly temperature measurements, recorded automatically, remained within 20±2°C. The mean light intensity calculated from a range of representative positions, measured once during the study, was 6440 lux (by cosine receptor), with a range of ±13%. The salinity of the test solutions, at the start and end of the test, was 0 to 0.1‰.

Table 5 Test solution pH and salinity measurements

  Nominal loading concentration of mixed ash (mg L-1)  Salinity %     pH      
   0 hours  72 hours  0 hours     72 hours
       Before adjustment  After adjustment  
 Control  -  -  7.4  -  7.6
 3.2  0.1  0.1  7.7  -  7.6
 5.6  -  -  8.5  7.5  7.7
 10  -  -  9.2  7.5  7.6
 18  0.1  0.1  8.5  7.6  7.6
 32  -  -  9.9  7.6  7.6
 56  0.1  0.1  10.2  7.5  7.6
Validity criteria fulfilled:
yes
Conclusions:
The effect concentrations (EC50) for yield and growth were found to be 8.5 mg/L and 21.3 mg/L, respectively
Executive summary:

Short-term toxicity of Ash toward a green alga Pseudokirchneriella subcapitata was studied in a GLP compliant laboratory according to the OECD Guideline 201 (Alga, Growth Inhibition Test). The 72 h test was performed at six concentrations of filtered Ash solutions with three replicates in constant illumination and at temperature of 22 ± 2°C. The nominal cell density was 0.5 x 104 cells. The growth curve for control was exponential. In the test, significant effects (p = 0.05) on alga growth was observed. The effect concentrations for yield and growth were found to be EyC50 = 8.5 mg/L and ErC50 = 21.3 mg/L, respectively.

Description of key information

Toxicity of Ash to Green alga Pseudokirchneriella subcapitata was determined in a GLP compliant laboratory study according to OECD Guideline 201 (Alga, Growth Inhibition Test ). The EC50 (72h) for growth rate was 21.3 mg/L and for yield it was 8.5 mg/L. 

Key value for chemical safety assessment

EC50 for freshwater algae:
21.3 mg/L

Additional information

Due to low water solubility of Ash, Water Accommondated Fractions of the test solutions were prepared.