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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:
5 February 2013 to 8 February 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
At test initiation and test termination (72 hours), duplicate 100 mL samples were taken and analysed for the test material. Samples analysed at 0 hours were removed from the test solutions in the volumetric flasks prior to filling the individual test flasks. Samples analysed at 72 hours were removed from composite solutions of replicate vessels for each treatment and the controls. Replicates A and B were frozen and stored at the test laboratory until it was delivered to the analytical laboratory for analysis.
Storage stability samples were prepared at 18, 100 and 320 mg/L, stored and analysed along with the exposure solutions to identify if there were any effects during the transportation of the exposure solutions.
Vehicle:
no
Details on test solutions:
Exposure solutions were prepared individually. The test material was weighed out, transferred to a 1000 mL volumetric flask and made to volume with test media. Test solutions were prepared with nominal concentrations of 18, 32, 56, 100, 180 and 320 mg/L.
The solutions were left to stir overnight for approximately 20 hours. On the day of testing the stirring was stopped and the solutions were left to stand for approximately 4 hours. The exposure solutions were transferred to an intermediate glass beaker using a syphon from the middle of the flasks to avoid transferring any settled test material. The solutions appeared clear at all concentrations.
Additionally, untreated AAP medium was used to prepare the control solution.
100 mL of the appropriate test solution was then placed in each of three replicate flasks for all of the treatment levels and six replicates for the controls. All test vessels were closed with foam bungs which permitted gas exchange.
In order to estimate the impact that the presence of algal biomass had on the test material concentration, an additional replicate flask (referred to as the uptake replicate) of the 100 mg/L treatment level was prepared. This flask, which was not inoculated with algae, was analysed at 72 hours of exposure for test material concentration. The results of these analyses were compared with the results for the 100 mg/L solution containing algae.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Freshwater green alga
- Strain: CCAP 278/4, Class Chlorophyceae
- Source (laboratory, culture collection): Culture collection of Algae and Protozoa, and was maintained in stock culture at the test laboratory

ACCLIMATION
Stock cultures were grown in 250 mL glass flasks each containing 100 mL of medium (AAP - Algal Assay Procedure medium). The flasks were plugged with foam stoppers which permitted gas exchange.
The stock cultures were maintained within the following conditions: a shaking rate of 100 ± 10 rpm, a temperature of 23 ± 2 °C and continuous illumination at the surface of the medium with an intensity range of 60 to 120 µE m^-2s^-1. Culture flasks were agitated continuously on an orbital shaker. Temperature was controlled using a Weiss Gallenkamp incubator.
The inoculum used to initiate the toxicity test with the test material was taken from a stock culture that had been transferred to fresh medium three days before testing.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
Not reported
Test temperature:
22.6 - 23.9 °C
pH:
6.75 - 6.99 at test initiation
8.44 - 8.56 at test termination
Dissolved oxygen:
Not reported
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentrations of 18, 32, 56, 100, 180 and 320 mg/L.

At 0 hours the measured concentrations ranged from less than the limit of detection (LOD), assessed as 0.04 mg/L, to 5.09 mg/L. At 72 hours the measured concentrations ranged from less than the LOD to 0.04 mg/L. Following this, all results are based on geometric mean measured concentrations.

Geometric mean concentrations: <0.04, <0.04, <0.07, <0.18, <0.26 and 0.45 mg/L for the nominal dose levels of 18, 32, 56, 100, 180 and 320 mg/L, respectively.
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL flasks. The test was conducted in an incubator designed to maintain the test temperature of 23 ± 2 °C, and an orbital shaker table provided a shaking rate of approximately 100 rpm.
- Type (delete if not applicable): Closed. All 100 mL test vessels were closed with foam bungs which permitted gas exchange.
- Initial cell density: approximately 1.0 x 10⁴ cells/mL
- Control end cells density: 90.4 x 10⁴ cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
Following preparation of the test solutions, 0.319 mL of an inoculum of Pseudokirchneriella subcapitata cells, at a density of 31.9 x 10⁵ cells/mL, was aseptically introduced into each 100 mL flask. This inoculum provided the required initial cell density of approximately 1.0 x 10⁴ cells/mL.

GROWTH MEDIUM
- Standard medium used: yes.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: AAP medium was prepared with sterile, demineralised water.
- Pesticides, polychlorinated biphenyls and toxic metals: Analysis for pesticides, PCBs and toxic metals in the dilution water revealed all levels to be within the defined acceptable limits.
- Culture medium different from test medium: No
- Intervals of water quality measurement: Temperature was measured continuously with a VWR minimum/maximum thermometer located in a flask of water adjacent to the test flasks in the incubator. Minimum and maximum temperatures were recorded daily.
The pH of the test solutions was measured at test initiation and at the termination of the 72-hour exposure period. Measurements at test initiation were conducted on the test solution remaining after the individual test flasks had been filled. At test termination, after cell counts were completed, the replicate solutions for each treatment and the control were respectively composited for pH measurements. Test solution pH was measured with a VWR pH100 meter.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: If necessary, the pH of the culture medium was adjusted to pH 7.58 ± 0.1 with either 0.1 M hydrochloric acid or 0.1 M sodium hydroxide.
- Photoperiod: Continuous light intensity photosynthetically-active radiation (PAR) range of 60 to 120 µE m^-2s^-1.
- Light intensity and quality: PAR of the test area was measured at test initiation and each 24 hour interval using PAR Quantum sensor developed by Skye Instruments Ltd.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
At each subsequent 24 hour interval, a single cell count was conducted on each replicate solution of the treatment levels and the controls using a haemocytometer (Neubauer Improved) and a compound microscope. One sample was removed from each flask for counting. One or more haemocytometer fields, each 0.10 x 0.10 cm in surface area and 0.010 cm deep and containing 0.00010 mL of culture, were examined for each sample until at least 400 algal cells or four fields were counted. Observations of the health of the algal cells were made at each 24-hour interval.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: ~1.8
- Range finding study: Yes
- Test concentrations in the range-finder: Nominal test material concentrations of 0.1, 1.0, 10 and 100 mg/L, and a control.
- Results used to determine the conditions for the definitive study: Yes. Two exposure vessels were established for each concentration and the control. At test termination, cells exposed to the control 0.01, 0.1 and 10 mg/L treatment level, were observed to be normal. On average, 15 % of cells at 100 mg/L were observed to be clumped together when compared to the control. Following 72 hours of exposure, cell densities in the 0.1, 1.0, 10, and 100 mg/L treatment levels averaged 119.6, 108.8, 110.9 and 29.4 x 10⁴ cells/mL, respectively. The control averaged 110.1 x 10⁴ cells/mL. Based on these results nominal concentrations of 18, 32, 56, 100, 180 and 320 mg/L were assigned to the definitive test.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.45 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
< 0.26 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
< 0.351 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95 % CL 0.3045 - 0.3854 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
< 0.26 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
BIOLOGICAL RESULTS
The 72 hour cell density in the control averaged 90.4 x 10⁴ cells/mL. Cell density in the 18, 32, 56, 100, 180 and 320 mg/L treatment levels averaged 88.8, 87.3, 96.7, 70.8, 82.1 and 17.8 x 10⁴ cells/mL, respectively.

Based on the results of Shapiro-Wilks' and Bartlett's test for cell biomass, expressed as yield, the 72 hour data set passed the requirements for normality and homogeneity of variance, therefore the 72 hour NOEC was determined by Bonferroni’s adjusted t- test. At 72 hours, no significant reduction in yield was detected in the <0.07, <0.18 and <0.26 mg/L treatment levels compared to the control data. Therefore, the 72 hour NOEC for yield was determined to be <0.26 mg/L. The 72-hour EyC10, EyC20 and EyC50 were calculated as <0.1281 mg/L with 95 % confidence intervals of N/A - 0.4231; <0.2642 mg/L with 95 % confidence intervals of 0.009454 - 0.3243; and <0.3511 mg/L with 95 % confidence intervals of 0.3045 and 0.3854, respectively.

Based on the results of Shapiro-Wilks’ and Bartlett’s tests for growth rates, the 72 hour data set passed the requirements for normality and homogeneity of variance, therefore, the 72 hour NOEC was determined by Bonferroni’s adjusted t- test. At 72 hours, no significant reduction in growth rate was detected in the <0.07, <0.18 and <0.26 mg/L treatment levels compared to the control data. Therefore, the 72 hour NOEC for growth rate was determined to be <0.26 mg/L. The 72 hour ErC10, ErC20 and ErC50 were calculated as <0.2901 mg/L with 95 % confidence intervals of 0.2443 - 0.3201; <0.3478 mg/L with 95 % confidence intervals of 0.3104 - 0.3781; and >0.45 mg/L, respectively.

EVALUATION OF TEST CONDITIONS
The pH of the test solutions ranged from 6.75 to 6.99 at test initiation. At test termination, the test solution pH ranged from 8.44 to 8.56. The increase in pH during the exposure is common in static algal tests and is due to photosynthesis by the algae. Continuous temperature monitoring established that the temperature ranged from 22.6 to 23.3 °C during the test period. The PAR (photosynthetically active radiation) of the test area ranged from 62.9 to 81.7 µE m^-2 s^-1 during the 72 hour exposure period.

ANALYTICAL RESULTS
At 0 hours the measured concentrations ranged from less than the limit of detection (LOD), assessed as 0.04 mg/L, to 5.09 mg/L. At 72 hours the measured concentrations ranged from less than the LOD to 0.04 mg/L.

VALIDITY CRITERIA
The following acceptance criteria were required by the study protocol: the cell growth in the control must increase from the initial density (1.0 x 10⁴ cells/mL) by more than 16 times after 72 hours of growth. Additionally, the mean coefficient of variation (CV) for section-by-section specific growth rates (day 0 to 1, 1 to 2 and 2 to 3) in the control replicates should not exceed 35 %. The CV for the average growth rate of the control for the entire test period (0 to 72 hour growth rate) should not exceed 7 %.
During this study, the 72-hour cell density in the control was 90.4 x 10⁴ cells/mL, the mean daily CV for growth rates was 19.7 % and the CV for 0 to 72 hour growth rate was 1.14 %, which all meet the above criteria.
Results with reference substance (positive control):
A reference inhibition study using 3, 5-Dichlorophenol, was performed on P. subcapitata at the test laboratory in December 2012. During this study, P. subcapitata were exposed to 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L of 3, 5-Dichlorophenol for 72 hours. After 72 hours of exposure, cell densities in the 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L treatment levels averaged 86.4, 76.8, 36.3, 2.4 and 1.3 x 10⁴ cells/mL, respectively. The control averaged 85.9 x 10⁴ cells/mL. Based on these results the 72-hour EC50 for growth rate and yield were calculated as 1.36 and 0.91 mg/L, respectively.
Reported statistics and error estimates:
Based on the results of statistical analysis performed for 72-hour total yield and average growth rate, the No-Observed-Effect Concentration (NOEC), the highest test concentration which demonstrated no statistically adverse effect (p ≤ 0.05) when compared to the control data, was determined. The data was first checked for normality using Shapiro-Wilks' Test (Weber et al., 1989) and for homogeneity of variance using Bartlett's Test (Horning and Weber, 1985). The NOEC values for both the growth rate and yield data were determined by Bonferroni’s adjusted t-test.
The 72-hour EC values were calculated for average growth rate (ErC10, ErC20 and ErC50 values) and yield (EyC10, EyC20 and EyC50 values) by linear interpolation of response (percent reduction of yield and growth rate as compared with the control). The EC10, EC20 and EC50 values are defined as the concentration of test material which caused a 10, 20 or 50 % reduction, respectively, in average growth rate or yield, compared to the control data. CETIS™ was used to perform all these statistical calculations.
Due to achieved concentration analysis demonstrating that nominal concentrations of 18 and 32 mg/L were below the LOD of the analytical method, these values have been excluded from the ECx calculations to avoid unrealistic bias on the data.

Table 3: Calculated growth rates of Pseudokirchneriella subcapitata after 24, 48 and 72 hours of exposure to the test material

Test

Concentration

(mg/L)

 

Growth rate (days-1)b

72 Hour %

Inhibitiona

Observation Interval (Hours)

0 - 24

0 - 48

0 - 72

Control

1

2

3

4

5

6

Mean

(SD)

1.852

1.852

1.786

1.636

1.236

1.675

1.673

(0.23)

1.461

1.591

1.586

1.395

1.514

1.586

1.522

(0.08)

1.481

1.513

1.495

1.514

1.483

1.521

1.501

(0.02)

NA

<0.04

1

2

3

Mean

(SD)

1.594

1.457

1.505

1.519

(0.07)

1.551

1.530

1.514

1.532

(0.02)

1.481

1.484

1.521

1.495

(0.02)

0

<0.04

1

2

3

Mean

(SD)

1.406

1.636

1.353

1.465

(0.15)

1.485

1.402

1.557

1.481

(0.08)

1.509

1.535

1.413

1.486

(0.06)

1

<0.07

1

2

3

Mean

(SD)

1.102

1.353

1.551

1.335

(0.22)

1.436

1.546

1.442

1.475

(0.06)

1.507

1.447

1.599

1.518

(0.08)

-1

<0.18

1

2

3

Mean

(SD)

1.675

1.406

1.027

1.37

(0.33)

1.525

1.310

1.442

1.426

(0.11)

1.356

1.489

1.400

1.415

(0.07)

6

<0.26

1

2

3

Mean

(SD)

1.551

1.457

1.296

1.435

(0.13)

1.520

1.416

1.388

1.441

(0.07)

1.469

1.532

1.393

1.464

(0.07)

2

0.45

1

2

3

Mean

(SD)

1.852

0.000

0.758

0.87

(0.93)

1.284

1.048

1.350

1.227

(0.16)

0.891

0.986

0.990

0.956

(0.06)

36

aPercent inhibition relative to the control

bMean, standard deviation (SD) and percent inhibition were calculated from original raw data, not from the rounded values presented in this table

NA = Not Applicable

Validity criteria fulfilled:
yes
Conclusions:
The 72-hour ErC50 value of the test material was calculated to be >0.45 mg/L (geometric mean concentration). The NOEC for growth rate was calculated as <0.26 mg/L (geometric mean concentration).
Executive summary:

The acute toxicity of the test material to the freshwater green alga Pseudokirchneriella subcapitata was investigated in a study conducted in accordance with the standardised guideline OECD 201 under GLP conditions.

The algae were exposed to the test material for 72 hours under static conditions at nominal concentrations of 18, 32, 56, 100, 180 and 320 mg/L. A control group was exposed to the Algal Assay Procedure (AAP) medium under the same conditions. The 0 to 72 hour biomass was expressed as yield and average growth rate relative to the performance of the control data.

This was a limit of solubility test as the test material was not soluble in water. The nominal solutions were prepared and stirred overnight and allowed to stand for approximately 4 hours. The solution used in the test was transferred using a syphon to avoid the transference of any settled test material.

At 0 hours the measured concentrations ranged from less than the limit of detection (LOD), assessed as 0.04 mg/L, to 5.09 mg/L. At 72 hours the measured concentrations ranged from less than the LOD to 0.04 mg/L. Following this, all results are based on geometric mean measured concentrations. The geometric mean concentrations were <0.04, <0.04, <0.07, <0.18, <0.26 and 0.45 mg/L for the nominal dose levels of 18, 32, 56, 100, 180 and 320 mg/L, respectively.

The 72 hour cell density in the control averaged 90.4 x 10⁴ cells/mL. Cell density in the 18, 32, 56, 100, 180 and 320 mg/L treatment levels averaged 88.8, 87.3, 96.7, 70.8, 82.1 and 17.8 x 10⁴ cells/mL, respectively.

At 72 hours, no significant reduction in yield was detected in the <0.07, <0.18 and <0.26 mg/L treatment levels compared to the control data. Therefore, the 72-hour NOEC for yield was determined to be <0.26 mg/L. The 72-hour EyC50 was calculated as <0.3511 mg/L with 95 % confidence intervals of 0.3045 and 0.3854.

At 72 hours, no significant reduction in growth rate was detected in the <0.07, <0.18 and <0.26 mg/L treatment levels compared to the control data. Therefore, the 72 hour NOEC for growth rate was determined to be <0.26 mg/L. The 72 hour ErC50 was calculated as >0.45 mg/L.

Under the conditions of this study, the 72-hour ErC50 value of the test material was calculated to be >0.45 mg/L (geometric mean concentration). The NOEC for growth rate was calculated as <0.26 mg/L (geometric mean concentration).

Description of key information

Study conducted to recognised testing guideline with GLP certification.

Key value for chemical safety assessment

Additional information

The acute toxicity of the test material to the freshwater green alga Pseudokirchneriella subcapitata was investigated in a study conducted in accordance with the standardised guideline OECD 201 under GLP conditions.

The algae were exposed to the test material for 72 hours under static conditions at nominal concentrations of 18, 32, 56, 100, 180 and 320 mg/L. A control group was exposed to the Algal Assay Procedure (AAP) medium under the same conditions. The 0 to 72 hour biomass was expressed as yield and average growth rate relative to the performance of the control data.

This was a limit of solubility test as the test material was not soluble in water. The nominal solutions were prepared and stirred overnight and allowed to stand for approximately 4 hours. The solution used in the test was transferred using a syphon to avoid the transference of any settled test material.

At 0 hours the measured concentrations ranged from less than the limit of detection (LOD), assessed as 0.04 mg/L, to 5.09 mg/L. At 72 hours the measured concentrations ranged from less than the LOD to 0.04 mg/L. Following this, all results are based on geometric mean measured concentrations. The geometric mean concentrations were <0.04, <0.04, <0.07, <0.18, <0.26 and 0.45 mg/L for the nominal dose levels of 18, 32, 56, 100, 180 and 320 mg/L, respectively.

The 72 hour cell density in the control averaged 90.4 x 10⁴ cells/mL. Cell density in the 18, 32, 56, 100, 180 and 320 mg/L treatment levels averaged 88.8, 87.3, 96.7, 70.8, 82.1 and 17.8 x 10⁴ cells/mL, respectively.

At 72 hours, no significant reduction in yield was detected in the <0.07, <0.18 and <0.26 mg/L treatment levels compared to the control data. Therefore, the 72-hour NOEC for yield was determined to be <0.26 mg/L. The 72-hour EyC50 was calculated as <0.3511 mg/L with 95 % confidence intervals of 0.3045 and 0.3854.

At 72 hours, no significant reduction in growth rate was detected in the <0.07, <0.18 and <0.26 mg/L treatment levels compared to the control data. Therefore, the 72 hour NOEC for growth rate was determined to be <0.26 mg/L. The 72 hour ErC50 was calculated as >0.45 mg/L.

Under the conditions of this study, the 72-hour ErC50 value of the test material was calculated to be >0.45 mg/L (geometric mean concentration). The NOEC for growth rate was calculated as <0.26 mg/L (geometric mean concentration).

The study was conducted to GLP and a standardised guideline. It was therefore assigned a reliability score of 1 and considered suitable for assessment as an accurate reflection of the test substance.

The available data are considered to be complete; the results determined, 72 h ErC50 >0.45 mg/L; 72 h NOEC <0.26 mg/L, were taken forward for risk assessment.