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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:

16 March 2020 - 09 December 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

(EC) 761/2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Concentrations: 1.0, 3.2, 10, 32 and 100 mg/L loading rates
Sampling method: 10 mLof the sample at 0 and 72 hours

Sample storage conditions before analysis was not reported

Vehicle:

no

Remarks:

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the study the test medium was prepared as a WAF of the test item.

Details on test solutions:

METHOD - PREPARATION AND APPLICATION OF TEST SOLUTION
Nominal amounts of test item (10, 32, 20, 64 and 200 mg) were each separately added to the surface of 10, 10, 2, 2, and 2 liters of culture medium to give the 1.0, 3.2, 10, 32 and 100 mg/L loading rates respectively. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour.

Visual observations made on the WAFs indicated that dispersed test item was present in the water column in the 10, 32 and 100 mg/L loading rates and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug. A 10 mL pippette, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75 to 100 mL discarded) to give the 1.0, 3.2, 10, 32 and 100 mg/L loading rate WAFs. Further filtration through one sheet of filter paper was required to remove as much undissolved test item as possible. Microscopic observations of the WAFs were performed after filtering and showed no micro-dispersions of test item to be present.

An aliquot (500 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (2.7 mL) to give an initial nominal cell density of 5.00 x 10^3 cells/mL.

Each loading rate WAF was inverted several times to ensure adequate mixing and homogeneity.


VALIDATION OF MIXING PERIOD
Preliminary investigational work was carried out to determine whether stirring for a prolonged period produced significantly higher levels of dissolved test item in the WAF. A WAF of a nominal loading rate of 100 mg/L was prepared in duplicate in ASTM and stirred using a stirring rate such that a vortex was formed to give a dimple at the water surface. One loading rate was stirred for a period of 23 hours and the other for a period of 95 hours. After a 1-Hour standing period and filtration through glass wool plug and filter paper, the mixtures were then removed by siphon and samples taken for chemical analysis.
It was evident from this work that increasing the stirring period did not significantly increase the amount of dissolved test item in the WAF and so preparation of the WAF was maintained at 24 hours.


RANGE-FINDING TEST
The loading rates to be used in the definitive test were determined by a preliminary range-finding test.
The range-finding test was conducted by exposing Raphidocelis subcapitata cells to nominal loading rates of 1.0, 10 and 100 mg/L for a period of 72 hours.
The test was conducted in 250 mL glass conical flasks each containing 100 mL of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were prepared for each control and test concentration.
Nominal amounts of test item (10, 20 and 200 mg) were each separately added to the surface of 10, 2 and 2 liters of culture medium to give the 1.0, 10 and 100 mg/L loading rates respectively. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour.

Visual observations made on the WAFs indicated that dispersed test item was present in the water column in the 10 and 100 mg/L loading rates and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75 to 100 mL discarded) to give the 1.0, 10 and 100 mg/L loading rate WAFs. Further filtration through one sheet of filter paper was required to remove as much undissolved test item as possible. Microscopic observations of the WAFs were performed after filtering and showed no micro-dispersions of test item to be present.

An aliquot (500 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (2.0 mL) to give an initial nominal cell density of 5.00 x 10^3 cells/mL. The control group was maintained under identical conditions but not exposed to the test item. Each loading rate WAF was inverted several times to ensure adequate mixing and homogeneity.
At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron® incubator) at 24 ±1 ºC under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 to 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter.
A sample of each loading rate WAF was taken for immediate chemical analysis at 0 and 72 hours in order to determine the stability of the test item under test conditions. A duplicate set of samples was taken on each occasion and stored frozen for further analysis if required.


INITIAL EXPERIMENT
Based on the results of the range-finding test the following loading rates were assigned to the definitive test: 0.50. 1.6, 5.0, 16 and 50 mg/L.
A 46% inhibition in growth rate was observed at a nominal test concentration of 50 mg/L and therefore failed to determine the EC50 value.
The results from the initial experiment were not used for reporting purposes.


DEFINITIVE TEST
Based on the results of the range-finding test and the initial experiment, the following loading rates were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100 mg/L.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
The test was carried out using Raphidocelis subcapitata strain CCAP 278/4. Liquid cultures of Raphidocelis subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.

ACCLIMATION
Approximately 3 to 4 days before the start of the test, inoculum cultures of algae was set up at an initial cell density of approximately 103 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker
(approximately 150 rpm) and constant illumination at 24 ±1 °C until the algal cell density was approximately 105 to 106 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Remarks on exposure duration:

According to OECD guidelines

Post exposure observation period:

No post exposure observation

Hardness:

Not reported

Test temperature:

24 ±1 °C

pH:

7.5 ± 0.1

Dissolved oxygen:

Not reported

Salinity:

N/A - freshwater

Conductivity:

N/A - freshwater

Nominal and measured concentrations:

Water Accommodated Fractions (WAFs) of the test item with nominal loading rates of 1.0, 3.2, 10, 32 and 100 mg/L

Details on test conditions:

TEST SYSTEM
Test vessel: 250 mL glass conical flasks. The flasks were plugged with polyurethane foam bungs
Type: closed
Fill volume: 100 mL
Initial cells density: Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 9.32 x 10^5 cells per mL. Inoculation of 500 mL of test medium with 2.7 mL of this algal suspension gave an initial nominal cell density of 5.00 x 10^3 cells per mL and had no significant dilution effect on the final test concentration.
Control end cells density: 4.65 x 10^5 cells per mL

No. of vessels per concentration: 3 replicates
No. of vessels per control: 3 replicates
No. of vessels per vehicle control: 3 replicates

GROWTH MEDIUM
Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
NaNO3 25.5 mg/L
MgCl2.6H2O 12.16 mg/L
CaCl2.2H2O 4.41 mg/L
MgSO4.7H2O 14.6 mg/L
K2HPO4 1.044 mg/L
NaHCO3 15.0 mg/L
H3BO3 0.186 mg/L
MnCl2.4H2O 0.415 mg/L
ZnCl2 0.00327 mg/L
FeCl3.6H2O 0.160 mg/L
CoCl2.6H2O 0.00143 mg/L
Na2MoO4.2H2O 0.00726 mg/L
CuCl2.2H2O 0.000012 mg/L
Na2EDTA.2H2O 0.30 mg/L
The culture medium was prepared using reverse osmosis purified deionized water* and the pH adjusted to 7.5 ± 0.1with 0.1N NaOH or HCl.

OTHER TEST CONDITIONS
- Adjustment of pH: pH adjusted to 7.5 ± 0.1with 0.1N NaOH or HC
- Light intensity and quality: continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 to 730 nm)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Determination of cell concentrations: Coulter® Multisizer Particle Counter. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.


TEST CONCENTRATIONS
Based on the results of the range-finding test the following loading rates were assigned to the definitive test: 0.50. 1.6, 5.0, 16 and 50 mg/L.
A 46% inhibition in growth rate was observed at a nominal test concentration of 50 mg/L and therefore failed to determine the EC50 value.
The results from the initial experiment were not used for reporting purposes.

Based on the results of the range-finding test and the initial experiment, the following loading rates were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100 mg/L.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EL10

Remarks:

ErL10 (loading rate WAF)

Effect conc.:

13 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL20

Remarks:

ErL20 (loading rate WAF)

Effect conc.:

29 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EL50

Remarks:

ErL50 (loading rate WAF)

Effect conc.:

> 100 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL10

Remarks:

EyL10 (loading rate WAF)

Effect conc.:

6.2 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: Inhibition of yield

Duration:

72 h

Dose descriptor:

EL20

Remarks:

EyL20 (loading rate WAF)

Effect conc.:

9.6 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: Inhibition of yield

Duration:

72 h

Dose descriptor:

EL50

Remarks:

EyL50 (loading rate WAF); 95% confidence limits 15 to 32 mg/L loading rate WAF

Effect conc.:

22 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: Inhibition of yield

Details on results:

Validation of Mixing Period
Preliminary investigational work (see Annex 4) indicated that there was no significant increase in the amount of dissolved test item when the preparation period was extended for longer than 24 hours. Therefore, for the purpose of testing the WAF was prepared using a stirring period of 23 hours followed by a 1-Hour settlement period.

Range-finding Test
The cell densities and percentage inhibition of growth values from the exposure of Raphidocelis subcapitata to the test item during the range-finding test are given in Table 1 (appended to attached background material in ‘Tables’). Based on this information loading rates of 1.6, 5.0, 16 and 50 mg/L were selected for the initial test.
Chemical analysis of the test preparations at 0 and 72 hours (see Annex 5, appended to attached background material) showed measured test item concentrations to range from 0.0068 to 108 mg/L at 0 hours to 0.027 to 110 mg/L at 72 hours indicating that the test item was stable under test conditions.

Initial Experiment
An initial experiment failed to determine the EC50. Therefore, based on the results of the initial experiment the following test concentrations were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100 mg/L LR WAF.

DEFINITIVE TEST
Chemical Analysis of Test Loading Rates
Due to a significant response in the original blank and control samples, the frozen duplicates were thawed and analysed with a fresh calibration standard and diluent, and the duplicate values used for reporting purposes. Chemical analysis of the test preparations (see Annex 5, appended to attached background material) at 0 hours showed measured test concentrations to range from 0.40 to 100 mg/L and from 0.34 to 99 mg/L at 72 hours.
The dissolved test item may have been one or several components of the test item. Given that toxicity cannot be attributed to a single component or mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

Growth Data
Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 2 (appended to attached background material in ‘Tables’). Daily specific growth rates for the control cultures are given in Table 3 (appended to attached background material in ‘Tables’).
Growth rate and yield values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 4 (appended to attached background material in ‘Tables’).
The mean cell densities versus time for the definitive test are presented in Figure 1 (appended to attached background material in ‘Figures’).
Percentage inhibition values are plotted against loading rate in Figure 2 and Figure 3 (appended to attached background material in ‘Figures’).
From the data given in Table 2 and Table 4 (appended to attached background material in ‘Tables’), it is clear that the growth rate (r) of Raphidocelis subcapitata (CCAP 278/4) was and yield were affected by the presence of the test item over the 72-Hour exposure period.
It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L.
Accordingly the following results were determined from the data:

Inhibition of Growth Rate
ErL10 (0 to 72 hour) : 13 mg/L loading rate WAF
ErL20 (0 to 72 hour) : 29 mg/L loading rate WAF
ErL50 (0 to 72 hour) : >100 mg/L loading rate WAF**
Where ErLx is the loading rate that reduced growth rate by x%.
Statistical analysis of the growth rate data was carried out for the control and and all loading rates using a Williams Multiple sequential t-test. There were no statistically significant differences (P≥0.05), between the control, 1.0, 3.2 and 10 mg/L loading rate WAF test groups; however, all other loading rates were significantly different (P<0.05) and, therefore the NOEL based on growth rate was 10 mg/L loading rate WAF. Correspondingly, the LOEL based on growth rate was 32 mg/L loading rate WAF.

Inhibition of Yield
EyL10 (0 to 72 hour) : 6.2 mg/L loading rate WAF
EyL20 (0 to 72 hour) : 9.6 mg/L loading rate WAF
EyL50 (0 to 72 hour) : 22 mg/L loading rate WAF; 95% confidence limits
15 to 32 mg/L loading rate WAF
Where EyLx is the loading rate that reduced yield by x%.
Statistical analysis of the yield data was carried out as in Section 6.4.2. There were no statistically significant differences (P≥0.05), between the control, 1.0, 3.2 and 10 mg/L loading rate WAF test groups; however, all other loading rates were significantly different (P<0.05) and, therefore the NOEL based on yield was 10 mg/L loading rate WAF.
Correspondingly, the LOEL based on yield was 32 mg/L loading rate WAF.

**It was not possible to calculate the EL50 value from the data generated as this was higher than the maximum concentration tested

Validation Criteria
The following data show that the cell concentration of the control cultures increased by a factor of 93 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.
Nominal cell density of control at 0 hours : 5.00 x 10^3 cells per mL
Mean cell density of control at 72 hours : 4.65 x 10^5 cells per mL
The mean coefficient of variation for section by section specific growth rate for the control cultures was 8% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.
The coefficient of variation for average specific growth rate for the control cultures over the test period (0 to 72 hour) was 5% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Observations on Cultures
All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 1.0 and 3.2 mg/L loading rate WAF. Some slightly misshapen cells were observed to be present in the test cultures at 10 mg/L loading rate WAF, whilst cell debris was observed to be present in the test culture at 32 and 100 mg/L loading rate WAF.

Water Quality Criteria
The pH values of the control and each test concentration are given in Table 2 (appended to attached background material in ‘Tables’).
The pH value of the control cultures was observed to decrease from pH 8.0 at 0 hours to pH 7.6 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines. Temperature was maintained at 24 ±1 ºC throughout the test.

Vortex Depth Measurements
The vortex depth was recorded at the start and end of the mixing period and was observed to have formed a dimple at the media surface.

Observations on Test Item Solubility
Observations on the test media were carried out during the mixing and testing of the WAFs. At the start of mixing the 1.0 mg/L loading rate WAF was observed to have formed a clear colorless media column with a small amount of fine particles of test item visible dispersed throughout whilst the 3.2 mg/L loading rate WAF was observed to have formed a clear colorless media column with a lump of test item within the dimple and a small amount of fine particles of test item visible dispersed throughout. The 10, 32 and 100 mg/L loading rate WAFs were observed to have formed very slightly hazy dispersions, with fine or large particles of test item visible dispersed throughout, which was also floating at the surface of the 32 and 100 mg/L loading rate WAFs. After stirring, and following a 1-Hour standing period, the 1.0 and 3.2 mg/L loading rate WAFs were observed to have formed clear colorless media columns with no visible test item, whilst the 10, 32 and 100 mg/L loading rate WAFs were observed to have formed very slightly hazy, slightly hazy or hazy media columns.
Microscopic examination of the WAFs showed there to be no micro-dispersions of test item present in the 1.0 and 3.2 mg/L loading rate WAFs and visual examination of the 10, 32 and 100 mg/L loading rate WAFs showed dispersed test item to be present. Microscopic examination of the WAFs after filtration with glass wool and filter paper, showed there to be no micro-dispersions of test item present.
At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control, 1.0, 3.2 and 10 mg/L loading rate WAF test cultures were observed to be green dispersions, whilst the 32 mg/L loading rate WAF test cultures were observed to be slightly pale green dispersions and the 100 mg/L loading rate WAF test cultures were observed to be pale green dispersions.

Results with reference substance (positive control):

A positive control (Covance study number YY61NS) used potassium dichromate as the reference item at concentrations of 0.125, 0.25, 0.50, 1.0 and 2.0 mg/L. The positive control was conducted between 20 January 2020 and 06 March 2020.
Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.

Exposure of Raphidocelis subcapitata (CCAP 278/4) to the reference item gave the following results:
ErC50 (0 to 72 hour) : 1.2 mg/L; 95% confidence limits 1.0 to 1.3 mg/L
EyC50 (0 to 72 hour) : 0.51 mg/L; 95% confidence limits 0.45 to 0.58 mg/L
No Observed Effect Concentration based on growth rate: 0.25 mg/L
No Observed Effect Concentration based on yield: 0.125 mg/L
Lowest Observed Effect Concentration based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration based on yield: 0.25 mg/L

The results from the positive control with potassium dichromate were within the normal ranges for this reference item*.

* In-house data from the previous five studies shows a mean ErC50 value of 1.5 mg/L (standard deviation = 0.11) and a mean EyC50 value of 0.69 mg/L (standard deviation = 0.11).

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Raphidocelis subcapitata has been investigated over a 72-Hour period and gave the following results:

EL50 (mg/L Loading Rate WAF):
Growth Rate = >100+
Yield = 22


95% Confidence Limits (mg/L Loading Rate WAF)
Growth Rate = Not determined+
Yield = 15 - 32


No Observed Effect Loading Rate (NOEL) (mg/L)
Growth Rate = 10
Yield = 10

Lowest Observed Effect Loading Rate (LOEL) (mg/L)
Growth Rate = 32
Yield = 32

+ Not calculated as EL50 value greater than the maximum concentration tested.

Executive summary:

Introduction

A study was performed to assess the effect of the test item on the growth of the green alga Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata). The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" and Method C.3 of Commission Regulation (EC) 761/2009. Study conducted under GLP conditions.

 

Methods

Due to the low aqueous solubility and complex nature of the test item for the purposes of the study the test medium was prepared as a Water Accommodated Fraction (WAF) of the test item.

Following a preliminary range-finding test and initial test, Raphidocelis subcapitata was exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 1.0, 3.2, 10, 32 and 100 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ±1 °C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

 

Results

Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.40 to 100 mg/L and from 0.34 to 99 mg/L at 72 hours.  

Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

Exposure of Raphidocelis subcapitata to the test item gave the following results:

 

 

Response Variable	EL50 (mg/L Loading Rate WAF)	95% Confidence Limits (mg/L Loading Rate WAF)	No Observed Effect Loading Rate (NOEL) (mg/L)	Lowest Observed Effect Loading Rate (LOEL) (mg/L)
Growth Rate	>100+	Not determined+	10	32
Yield	22	15 - 32	10	32

Description of key information

The effect of the test item on the growth of Raphidocelis subcapitata has been investigated over a 72-Hour period and gave the following results:

EL50 (mg/L Loading Rate WAF):
Growth Rate = >100+
Yield = 22

95% Confidence Limits (mg/L Loading Rate WAF)
Growth Rate = Not determined+
Yield = 15 - 32

No Observed Effect Loading Rate (NOEL) (mg/L)
Growth Rate = 10
Yield = 10

Lowest Observed Effect Loading Rate (LOEL) (mg/L)
Growth Rate = 32
Yield = 32

+ Not calculated as EL50 value greater than the maximum concentration tested.

Additional information

Introduction

A study was performed to assess the effect of the test item on the growth of the green alga Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata). The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" and Method C.3 of Commission Regulation (EC) 761/2009. Study conducted under GLP conditions.

 

Methods

Due to the low aqueous solubility and complex nature of the test item for the purposes of the study the test medium was prepared as a Water Accommodated Fraction (WAF) of the test item.

Following a preliminary range-finding test and initial test, Raphidocelis subcapitata was exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 1.0, 3.2, 10, 32 and 100 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ±1 °C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

 

Results

Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.40 to 100 mg/L and from 0.34 to 99 mg/L at 72 hours.  

Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

Exposure of Raphidocelis subcapitata to the test item gave the following results:

 

 

Response Variable	EL50 (mg/L Loading Rate WAF)	95% Confidence Limits (mg/L Loading Rate WAF)	No Observed Effect Loading Rate (NOEL) (mg/L)	Lowest Observed Effect Loading Rate (LOEL) (mg/L)
Growth Rate	>100+	Not determined+	10	32
Yield	22	15 - 32	10	32