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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:
08 May 2012 - 08 November 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Testing performed according to OECD guideline 201 and was performed in compliance with UK GLP standards in accordance with the OECD Principles on Good Laboratory Practice.
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
conducted in sealed vessels
Qualifier:
according to
Guideline:
EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II)
Deviations:
yes
Remarks:
conducted in sealed vessels
Principles of method if other than guideline:
The test item was known to be volatile and hence testing was considered in completely filled, stoppered test vessels in order to minimize possible losses due to volatilization. Following the recommendations of published data (Herman et al 1990) in order to prevent inhibition of growth due to the restriction of gaseous exchange, additional sodium bicarbonate was added to the culture medium to provide a source of carbon dioxide for algal growth.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- Name of test material: Amyl Salicylate
- Substance type: Multi-Constituent Substance
- Physical state: clear colourless liquid
- Analytical purity: 99.99% (sum of isomers)
- Lot/batch No.: PE0022143
- Date received: 03-October-2012
- Expiration date of the lot/batch: 19-August-2012
- Storage condition of test material: room temperature in the dark
Analytical monitoring:
yes
Details on sampling:
- Concentrations:
Nominal Range Finding: 0.0037, 0.037, 0.37 and 3.7 mg/L
Nominal Definitive Test: 0.24, 0.47, 0.93, 1.9 and 3.7 mg/L
0-Hour Measured Definitive Test: 0.17, 0.27, 0.61, 1.3, and 2.7 mg/L
- Sampling: Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter Multiisizer Particle Counter.
- Sample storage conditions before analysis: samples were sealed and incubated at 24 +/- 1°C under continuous illumination and constantly shaken at approximately 150 rpm for 96 hours.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: An amount of test material (550mg) was dispersed in 11 litres of culture medium with the aid of propeller stirring at approximately1500rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved material was removed by filtration through a 0.2µm Sartorius Sartopore filter (first approximate 2 litres discarded to precondition the filter). to give a saturated solution with a 0-hour measured concentration of 2.7 mg/L. A series of dilutions was made from those saturated solution to give further stock solutions of 1.3, 0.61, 0.27 and 0.17 mg/L. An aliquot (1700mL) of each of the stock solutions was separately inoculated with 31 mL algal suspension to give the 0-hour measured concentrations of 0.17, 0.27, 0.61, 1.3, and 2.7 mg/L
The Stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity. Due to the possible light sensitive nature of the test item a;; test item preparation was performed under laboratory safety lighting/shielding from the light.
The concentration and stability of the test solutions were verified by chemical analysis at 0m, 24, 48 72 and 96 hours.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokichneriella subcapitata
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Post exposure observation period:
96 h
Hardness:
see culture medium below
Test temperature:
incubated at 24 +/- 1°C
pH:
pH adjusted to 7.5 ±0.1 with 0.1N NaOH or HCl.
Dissolved oxygen:
no data
Salinity:
see culture medium below
Nominal and measured concentrations:
Nominal Range Finding: 0.0037, 0.037, 0.37 and 3.7 mg/L
Nominal Definitive Test: 0.24, 0.47, 0.93, 1.9 and 3.7 mg/L
0-Hour Measured Definitive Test: 0.17, 0.27, 0.61, 1.3, and 2.7 mg/L
Details on test conditions:
Culture Medium

NaNO3 25.5 mg/L
MgCl2.H2O 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
H2BO3 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

Prepared using reverse osmosis purified deionised water, pH adjusted to 7.5 ±0.1 with 0.1N NaOH or HCl.

For the definitive test, additional sodium bicarbonate (500mg/L) was added to the prepared culture medium prior to use.
Reference substance (positive control):
yes
Remarks:
zinc chloride
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.77 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.:
0.53 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks on result:
other: 0.48-0.58
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.49 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 0.43-0.56
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.2 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.:
0.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.11 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
0.94 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
0.68 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks on result:
other: 0.63-0.74
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
0.65 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 0.59-0.72
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
0.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
0.24 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
0.24 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no abnormalities detected in the control or test cultures at 0.17, 0.27, 0.61 and 1.3mg/L, however few intact cells were observed to be present in the test concentration 2.7 mg/L.
- Unusual cell shape:
- Colour differences: At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 96-hour test period all control, 0.17, 0.27, and 0.61 mg/L test cultures were observed to be green dispersions whilst the 1.3 and 2.7 mg/L test cultures were observed to be clear colourless solutions.
- Other: A regrowth experiment was conducted which showed that the test item was algistatic in effect.
- Any observations that might cause a difference between measured and nominal values: The test item had shown a decline in measured concentration, further analysis showed that this was due to both adsorption to algal cells and some losses due to instability of the test item. It was therefore considered appropriate to base the results on the geometric mean measured test concentrations using the 72 and 96 hour results obtained from the samples prepared with the omission of algal cells as these reflect more accurately the concentrations of test item to which the algae were exposed. see the table in "any other information on results"
Results with reference substance (positive control):
72h EC50= 0.23 (growth) 0.090 (yield) 0.12 (biomass) mg/L
96h EC50= 0.30 (growth) 0.073 (yield) 0.096 (biomass) mg/L
72h NOEC= 0.010 (growth) 0.010 (yield) 0.032 (biomass) mg/L
96h NOEC= 0.010 (growth) 0.010 (yield) 0.010 (biomass) mg/L
Historical in house data for the exposure of Pseudokirchneriella subcapitata to zinc chloride using standard exposure conditions (100 mL of the test preparation in a 250 mL conical flask plugged with a polyurethane foam bung) gave EC50 values which were slightly higher, but NOEC values which were a factor of 10 higher than those observed.
It was evident from these results that the use of a modified test system to reduce losses of test item through volatility (completely filled and sealed test vessels) has a slight effect on the EC50 values obtained and, has a notable effect on the No Observed Effect Concentration (NOEC). As such it can be considered that the results obtained from the definitive test give a worst case result for the test item.

 


 





























































































































Time Point (hours)Response VariableMeasured:
initial / geometric mean
EC50 (mg/l)95% confidence limits (mg/L)No Observed Effect Concentration (NOEC) mg/L)Lowest Observed Effect Concentration (LOEC) (mg/L)
72Growth Rateinitial0.9*0.270.61
72Yieldinitial0.660.61-0.710.270.61
72Biomassinitial0.620.55-0.700.170.27
96Growth Rateinitial0.91*0.611.3
96Yieldinitial0.680.64-0.730.270.61
96Biomassinitial0.660.60-0.720.270.61
72Growth Rategeometric mean0.77*0.20.48
72Yieldgeometric mean0.530.48-0.580.20.48
72Biomassgeometric mean0.490.43-0.560.110.2
96Growth Rategeometric mean0.94*0.61.4
96Yieldgeometric mean0.680.63-0.740.240.6
96Biomassgeometric mean0.650.59-0.720.240.6
*=It was not possible to calculate 95% confidence limits for the ErC50 values as the data generated did not fit the models available for the calculation of the confidence limits.
Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 96 hour period and gave the results listed in the table above in "any other information on results" for 0-hour measured concentrations and geometric mean measured test concentrations.
A regrowth experiment was conducted which showed that the test item was algistatic in effect.
A positive control test showed that the use of a modified test system to reduce losses of test item through volatility (completely filled and sealed test vessels) had a slightly lowering effect on the EC50 values obtained and, has a notable effect on the No Observed Effect Concentration (NOEC). As such it can be considered that the results obtained from the definitive test give a worst case result for the test item.
Executive summary:

A study was performed to assess the effect of the test item on the growth of the green alga 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" referenced as method C3 of Commission Regulation (EC) No 761/2009 and the US EPA Draft Ecological Effects test guideline OPPTS 850.5400.


 


Methods
Pre-study solubility work conducted indicated that it was not Possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication.


A pre study media preparation trial indicated that a dissolved test item concentration of approximately 3.7 mg/L was obtained from a saturated Solution Method of Preparation indicating this to be the limit of water solubility of this item under test conditions.


Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to solutions of the test item at 0-hour measured concentrations of 0.17, 0.27, 0.61, 1.3 and 2.7 mg/L (three replicate flasks per concentration) for 96 hours, under constant illumination and shaking at a temperature of 24 ±1°C. The test item solutions were prepared by stirring an excess (50mg/L) of test item in culture medium using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period and undissolved test item was removed by filtration (0.2µm Sartorius Sartopore filter, first approximate 2 liters discarded in order to pre-condition the filter) to produce a saturated solution of the test item with a 0-hour measured concentration of 2.7mg/L. This saturated solution was then further diluted as necessary, to provide the remaining test groups.


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


The test item was known to be volatile and hence testing was considered in completely filled, stoppered test vessels in order to minimize possible losses due to volatilization. Following the recommendations of published data (Herman et al 1990) in order to prevent inhibition of growth due to the restriction of gaseous exchange, additional sodium bicarbonate was added to the culture medium to provide a source of carbon dioxide for algal growth.


 


The test item was known to be volatile (EPIWIN V3.20 predicted Henry’s law constant =1.2 Pa.m3/mol) and hence it was likely that losses of test item would occur during the time taken to prepare the required test concentrations or analysis. As such, samples were taken for immediate chemical analysis to minimize losses as far as practically possible. Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.17 to 2.7 mg/L.


Exposure of Pseudokirchneriella subcapitata to the test item based on the 0-hour measured test concentrations gave the following results:








































































Time Point (hours)



Response Variable



Measured:
initial / geometric mean



EC50 (mg/l)



95% confidence limits (mg/L)



No Observed Effect Concentration (NOEC) mg/L)



Lowest Observed Effect Concentration (LOEC) (mg/L)



72



Growth Rate



initial



0.9



*



0.27



0.61



72



Yield



initial



0.66



0.61-0.71



0.27



0.61



72



Biomass



initial



0.62



0.55-0.70



0.17



0.27



96



Growth Rate



initial



0.91



*



0.61



1.3



96



Yield



initial



0.68



0.64-0.73



0.27



0.61



96



Biomass



initial



0.66



0.60-0.72



0.27



0.61



*=It was not possible to calculate 95% confidence limits for the ErC50 values as the data generated did not fit the models available for the calculation of the confidence limits.



 


As analysis of the range finding test preparations showed a decline in measured test concentration it was considered appropriate to prepare additional test samples at the start of the test which were sent for chemical analysis at 24, 48 and 72 hours in addition to those provided at 96 hours.


Analysis of the test preparations at 24, 48, 72 and 96 hours showed a concentration dependent decline in measured test concentration with significant losses to below the limit of quantitation (LOQ) of the analytical method employed, which was determined to be 0.029 mg/L, being observed in the 0.24 and 0.47 mg/L, test sample at 96 hours.


As the decline in measured concentration observed in the range-finding test followed a concentration dependant pattern it was considered likely that the test item was absorbing to the algal cells present. Therefore, additional test samples were prepared at the start of the test with the omission of algal cells and incubated alongside the test. Analysis of these additional samples at 72 hours showed measured test concentrations in the range of 46% to 77% of the 0-hour measured concentrations were obtained. At 96 hours measured test concentrations in the range of 25% to 117% of the 0 hour measured concentrations were obtained.


These results would suggest that whilst the majority of the decline in measured concentration was due to adsorption of the test item to the algal cells present, some losses were occurring due to adsorption of the test item to the algal cells present, some losses were occurring due to instability of the test item. It was therefore considered appropriate to base the results on the geometric mean measured test concentrations using the 72 and 96 hour results obtained from the samples prepared with the omission of algal cells as these reflect more accurately the concentrations of the test item to which the algae were exposed.


Exposure of Pseudokirchneriella subcapitata to the test item gave the following results based on the geometric mean measured test concentrations.








































































Time Point (hours)



Response Variable



Measured:
initial / geometric mean



EC50 (mg/l)



95% confidence limits (mg/L)



No Observed Effect Concentration (NOEC) mg/L)



Lowest Observed Effect Concentration (LOEC) (mg/L)



72



Growth Rate



geometric mean



0.77



*



0.2



0.48



72



Yield



geometric mean



0.53



0.48-0.58



0.2



0.48



72



Biomass



geometric mean



0.49



0.43-0.56



0.11



0.2



96



Growth Rate



geometric mean



0.94



*



0.6



1.4



96



Yield



geometric mean



0.68



0.63-0.74



0.24



0.6



96



Biomass



geometric mean



0.65



0.59-0.72



0.24



0.6



*=It was not possible to calculate 95% confidence limits for the ErC50 values as the data generated did not fit the models available for the calculation of the confidence limits.



 


A regrowth test was performed which showed the test item to be algistatic in effect.


A positive control test showed that the use of a modified test system to reduce losses of test item through volatility (completely filled and sealed test vessels) had a slightly lowering effect on the EC50values obtained, and a notable effect on the No Observed Effect Concentration (NOEC) compared with that obtained in a conventional test system. As such it can be considered that the results obtained from the definitive test give a worst case result for the test item.

Description of key information

EC50(growth) = 0.77mg/L based on geometric mean measured concentration

Key value for chemical safety assessment

EC50 for freshwater algae:
0.77 mg/L
EC10 or NOEC for freshwater algae:
0.2 mg/L

Additional information

The test item was known to be volatile and hence testing was conducted in completely filled, stoppered test vessels in order to minimize possible losses due to volatilization. However, a positive control test showed that the use of a modified test system to reduce losses of test item through volatility (completely filled and sealed test vessels) had a slightly lowering effect on the EC50values obtained, and, a notable effect on the No Observed Effect Concentration (NOEC) compared with that obtained in a conventional test system. As such it can be considered that the results obtained from the definitive test give a worst case result for the test item.

The preferred observational endpoint in this study is algal growth rate inhibition because it is not dependent on the test design (see Guidance on information requirements and chemical safety assessment Chapter R.7b: Endpoint specific guidance v1.1). Thus the 72 hr EC₅₀ and NOEC based on growth rate are used for classification purposes.