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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June to July 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This robust summary has a reliability rating of 1 because the study followed a standard guideline, followed GLP guidelines, and was conducted without deviations that would invalidate the study.
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
The water accommodated fraction (WAF) of the test material was prepared by stirring the test material in the exposure solution for approximately 24 hours. The stirring was as vigorous as possible without causing an emulsion to form. After stirring, the WAF was allowed to settle for 1 hour before removing the aqueous phase for testing.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
Organisms used in the test were from a laboratory culture, originally derived from a strain (ATCC 22662) obtained from the American Type Culture Collection, Maryland, Ohio, USA.
Test type:
static
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
None
Test temperature:
24 to 26 degrees C
pH:
Within Guideline requirements
Nominal and measured concentrations:
The nominal loading rate was 1000 mg/L. A control was also tested.
Details on test conditions:
The study was conducted in sealed test systems with no headspace that were not renewed during the study. The test systems used were 300 ml glass Erlenmeyer flasks. The treatment level was evaluated in triplicate test systems and the control in 6 replicate test systems. Control and treatment systems were innoculated with a concentration of 5000 cells/ml. Test systems were incubated under constant illumination (approximately 3000 lux) on an orbital incubator.
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Reported statistics and error estimates:
The growth of algae in the the test systems was greater than the controls, which performed normally, therefore no analyses were required.
Validity criteria fulfilled:
yes
Conclusions:
Growth of alga cultures, as measured by biomass and growth rate, exposed to a water accommodated fraction of the test substance was not inhibited over 72 hours. The growth of algae in the the test systems was greater than the controls, which performed normally. Therefore, the 72-hr EL50 values for the two endpoints are reported as >1000 mg/L. The 72-hr NOELR values for biomass and growth rate are reported as 1000 mg/L, respectively.
Executive summary:

Growth of alga cultures, as measured by biomass and growth rate, exposed to a water accommodated fraction of the test substance was not inhibited over 72 hours. The growth of algae in the the test systems was greater than the controls, which performed normally. Therefore, the 72-hr EL50 values for the two endpoints are reported as >1000 mg/L. The 72-hr NOELR values for biomass and growth rate are reported as 1000 mg/L, respectively.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
June 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This robust summary has a reliability rating of 1 because the study followed a standard guideline, followed GLP guidelines, and was conducted without deviations that would invalidate the study.
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
The water accommodated fraction of the test material were prepared by stirring the test material in the exposure solution for 25 hours after which the the aqueous phase was removed for testing.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
Organisms used in the test were from a laboratory culture, originally derived from a strain (ATCC 22662) obtained from the American Type Culture Collection, Maryland, Ohio, USA.
Test type:
static
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
None
Test temperature:
24 to 26 degrees C
pH:
Within Guideline requirements
Nominal and measured concentrations:
The nominal loading rate was 1000 mg/L. A control was also tested.
Details on test conditions:
The study was conducted in sealed test systems with no headspace that were not renewed during the study. The test systems used were 300 ml glass Erlenmeyer flasks. The treatment level was evaluated in triplicate test systems and the control in 6 replicate test systems. Control and treatment systems were innoculated with a concentration of 5000 cells/ml. Test systems were incubated under constant illumination (approximately 3000 lux) on an orbital incubator.
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Reported statistics and error estimates:
The growth of algae in the the test systems was greater than the controls, which performed normally, therefore no analyses were required.
Validity criteria fulfilled:
yes
Conclusions:
Growth of alga cultures, as measured by biomass and growth rate, exposed to a water accommodated fraction of the test substance was not inhibited over 72 hours. The growth of algae in the the test systems was greater than the controls, which performed normally. Therefore, the 72-hr EL50 values for the two endpoints are reported as >1000 mg/L. The 72-hr NOELR values for biomass and growth rate are reported as 1000 mg/L, respectively.
Executive summary:

Growth of alga cultures, as measured by biomass and growth rate, exposed to a water accommodated fraction of the test substance was not inhibited over 72 hours. The growth of algae in the the test systems was greater than the controls, which performed normally. Therefore, the 72-hr EL50 values for the two endpoints are reported as >1000 mg/L. The 72-hr NOELR values for biomass and growth rate are reported as 1000 mg/L, respectively.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
The study was conducted between 14 August 2014 and 03 September 2014.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Vehicle:
no
Details on test solutions:
A nominal amount of test item (230 mg) was added to the surface of 2.3 litres of culture medium in a sealed vessel with minimal headspace to give the 100 mg/L loading rate. 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 mixture allowed to stand for 1 hour. 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. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 100 mg/L loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present.

An aliquot (2 litres) of the WAF was inoculated with algal suspension (10 mL) to give the required test concentration of 100 mg/L loading rate WAF.

Total Organic Carbon (TOC) analysis was performed on the test preparations at 0 and 72 hours.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the 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. The master cultures were maintained in the laboratory under constant aeration and illumination at 21 ± 1 °C.

Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 1E+03 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 1E+04 – 1E+05 cells/mL.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Nominal and measured concentrations:
Nominal: 10 and 100 mg/L
Details on test conditions:
Culture Medium
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture with the addition of 500 mg/L of sodium bicarbonate to counteract the increase in pH due to algal growth in an enclosed system (Herman et al 1990).

Procedure
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.

Previous studies conducted on similar test items (e.g. Harlan Study Number 41304080) indicated that a 23-Hour stirring period followed by a 1-Hour standing period was sufficient to ensure that the maximum dissolved test item concentration was obtained in a Water Accommodated Fraction (WAF).


Range-Finding Test
The loading rate to be used in the definitive test was determined by a preliminary range-finding test.

The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to nominal loading rates of 10 and 100 mg/L for a period of 72 hours.

The test was conducted in 250 mL glass conical flasks each completely filled with test preparation and sealed with a ground glass stopper to reduce evaporation and minimize losses due to the test items potentially volatile nature. Two replicate flasks were used for each control and test concentration.

Nominal amounts of test item (23 and 230 mg) were each separately added to the surface of 2.3 litres of culture medium in sealed vessels with minimal headspace to give the 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. 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. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 10 and 100 mg/L loading rate WAFs. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present.

An aliquot (1 litre) of each of the loading rate WAFs was separately inoculated with algal suspension (6.0 mL) to give the required test concentrations of 10 and 100 mg/L loading rate WAF.

The control group was maintained under identical conditions but not exposed to the test item.

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 sealed with ground glass stoppers and incubated (INFORS Multitron® Version 2 incubator) at 24 ± 1 ºC under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 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.


Definitive Test
Based on the result of the range-finding test a "limit test" was conducted at a single loading rate of 100 mg/L to confirm that no effect on algal growth was observed.

Experimental Preparation
A nominal amount of test item (230 mg) was added to the surface of 2.3 litres of culture medium in a sealed vessel with minimal headspace to give the 100 mg/L loading rate. 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 mixture allowed to stand for 1 hour. 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. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 100 mg/L loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present.

An aliquot (2 litres) of the WAF was inoculated with algal suspension (10 mL) to give the required test concentration of 100 mg/L loading rate WAF.

Total Organic Carbon (TOC) analysis was performed on the test preparations at 0 and 72 hours.


Exposure Conditions
As in the range-finding test 250 mL glass conical flasks were used. Six flasks each completely filled with test preparation were used for the control and 100 mg/L loading rate WAF treatment group.

The control group was maintained under identical conditions but not exposed to the test item.

Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 9.72E+05 cells per mL. Inoculation of 2 litres of test medium with 10 mL of this algal suspension gave an initial nominal cell density of 5E+03 cells per mL and had no significant dilution effect on the final test concentration.

The flasks were sealed with ground glass stoppers and incubated (INFORS Multitron® Version 2 incubator) at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

Evaluations
Test Organism Observations
Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.

Data Analysis
Comparison of Growth Rates
The average specific growth rate for a specified period is calculated as the logarithmic increase in biomass from the equation:

µ = (1n Nn – 1n N1) / (tn – t1)

Where:
µ = average specific growth rate from time t1 to tn
N1 = cell concentration at t1
Nn = cell concentration at tn
t1 = time of first measurement
tn = time of nth measurement

The average specific growth rate over the test duration was calculated for each replicate control and test item vessel using the nominally inoculated cell concentration as the starting value rather than the measured starting value in order to increase the precision of the calculation.

In addition the section by section specific growth rate (days 0-1, 1-2 and 2-3) was calculated for the control cultures and the results examined in order to determine whether the growth rate remained constant.

Percentage inhibition of growth rate for each replicate test item vessel was calculated using the following equation:

Ir = ((µc - µt) / µc) x 100

Where:
Ir = percentage inhibition of average specific growth rate
µc = mean average specific growth rate for the control cultures
µt = average specific growth rate for the test culture

Comparison of Yield
Yield is calculated as the increase in biomass over the exposure period using the following equation:

Y = Nn – N0

Where:
Y = yield
N0 = cell concentration at the start of the test
Nn = cell concentration at the end of the test

For each test concentration and control the mean value for yield along with the standard deviation was calculated. Percentage inhibition of yield was calculated using the following equation:

Iy = ((Yc – Yt) / Yc) x 100

Where:
Iy = percentage inhibition of yield
Yc = mean value for yield in the control group
Yt = mean value for yield for the treatment group


Determination of ELx Values
ELx values were determined by inspection of the growth rate and yield data after 72 hours.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
WAF loading rate
Basis for effect:
growth rate
Remarks on result:
other: The toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
WAF loading rate
Basis for effect:
growth rate
Details on results:
Range-finding Test
The results showed no effect on growth at 10 and 100 mg/L loading rate WAF.

Based on this information a single loading rate of six replicates of 100 mg/L was selected for the definitive test. This experimental design conforms to a "limit test" to confirm that no effect on growth was observed.


Definitive Test
Total Organic Carbon Analysis
Total Organic Carbon (TOC) analysis of the 100 mg/L loading rate WAF test preparations at 0 and 72 hours showed measured concentrations of less than the limit of quantification (LOQ), determined to be 1.0 mg C/L were obtained.


Growth Data
From the data it is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were not 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 - 72 h): >100 mg/L loading rate WAF
ErL20 (0 - 72 h): >100 mg/L loading rate WAF
ErL50 (0 - 72 h): >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 100 mg/L loading rate WAF test group using a Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955). There were no statistically significant differences (P0.05), between the control and 100 mg/L loading rate WAF test group and therefore the "No Observed Effect Loading Rate" (NOEL) based on growth rate was 100 mg/L loading rate WAF.

Inhibition of Yield
EyL10 (0 - 72 h): >100 mg/L loading rate WAF
EyL20 (0 - 72 h): >100 mg/L loading rate WAF
EyL50 (0 - 72 h): >100 mg/L loading rate WAF

Where EyLx is the loading rate that reduced yield by x%.

There were no statistically significant differences between the control and 100 mg/L loading rate WAF (P≥0.05), and, therefore the "No Observed Effect Loading Rate" (NOEL) based on yield was 100 mg/L loading rate WAF.
Results with reference substance (positive control):
A positive control (Harlan Study Number 41303826) used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L.

Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.

Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:

ErC50 (0 – 72 h): 1.1 mg/L; 95% confidence limits 0.91 – 1.2 mg/L
EyC50 (0 – 72 h): 0.51 mg/L; 95% confidence limits 0.45 – 0.59 mg/L

No Observed Effect Concentration (NOEC) based on growth rate: 0.25 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration (LOEC) based on yield: 0.50 mg/L

The results from the positive control with potassium dichromate were within the normal ranges for this reference item.
Reported statistics and error estimates:
A Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) was carried out on the growth rate and yield data after 72 hours for the control and the 100 mg/L loading rate to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

Validation Criteria

The following data show that the cell concentration of the control cultures increased by a factor of 118 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.

 

Mean cell density of control at 0 hours: 5.65E+03 cells per mL

Mean cell density of control at 72 hours: 6.65E+05 cells per mL

 

The mean coefficient of variation for section by section specific growth rate for the control cultures was 25% 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 – 72 h) was 3% 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. There were no abnormalities detected in any of the control or test cultures.

 

Water Quality Criteria

Temperature was maintained at 24 ± 1 ºC throughout the test.

 

The pH value of the control cultures was observed to increase from pH 7.9 at 0 hours to pH 9.7 at 72 hours. This increase was considered to be due to the amount of carbon dioxide required by the large number of algal cells in the log phase of growth exceeding the transfer rate of CO2from the gaseous phase to the aqueous phase. In this situation CO2required for photosynthesis and growth would be derived from bicarbonate in solution which results in an increase in the pH of the culture. The increase in pH after 72 hours was in excess of that recommended in the EC Guidelines (1.5 pH units after 72 hours). This was considered to have had no adverse effect on the results of the study given that the increase in cell concentration in the control cultures exceeded the validation criterion given in the Test Guidelines.

 

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 WAF.

 

At both the start and end of the mixing period, and following a 1-Hour standing period, the 100 mg/L loading rate WAF was observed to have formed a clear colorless media column with an oily globule of test item floating at the media surface. Microscopic examination of the WAF showed there to be no globules or 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 and test cultures were observed to be green dispersions.

Validity criteria fulfilled:
yes
Conclusions:
Measured toxicity data are available for Shell GTL Solvent GS190 (Hydrocarbons, C10-C13, n-alkanes, isoalkanes, <2% aromatics) to the freshwater green alga Pseudokirchneriella subcapitata. The test was conducted under static (no renewal of the test media) conditions in accordance with OECD Test Guideline 201 and method C3 of EC Regulation No. EC 761/2009. Appropriate modifications to the test and media preparation procedures were made to take account of the test substance containing multiple constituents, having low solubility in water and being potentially volatile. No effects on growth (expressed in terms of yield (y) and growth rate (r)) of P. subcapitata were observed after 72 hours exposure to the test medium prepared as a water-accommodated fraction (WAF) at a loading rate of 100 mg/l; 72-hour EyL50 and ErL50 values were >100 mg/l and NOELR values were ≥100 mg/l. Total Organic Carbon analysis of the 100 mg/l loading rate WAF test preparations at 0 and 72 hours showed measured carbon concentrations of less than the limit of quantification, determined to be 1.0 mg C/l. The results of the test are considered to be reliable.
Executive summary:

Introduction

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 C.3 of Commission Regulation (EC) 761/2009.

 

Methods

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

 

Following a preliminary range-finding test,Pseudokirchneriella subcapitatawas exposed to a Water Accommodated Fraction (WAF) of the test item, at a single nominal loading rate of 100 mg/L (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C.

 

Due to the potentially volatile nature of the test item, testing was conducted in completely filled, stoppered test vessels in order to minimize possible losses due to volatilization. Following the recommendations of published data (Hermanet al1990) 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.

 

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…….

Total Organic Carbon (TOC) analysis of the 100 mg/L loading rate WAF test preparations at 0 and 72 hours showed measured carbon concentrations of less than the limit of quantification (LOQ), determined to be 1.0 mg C/L were obtained.

 

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 Pseudokirchneriella subcapitata to the test item gave EL50values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.

 

It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L loading rate WAF.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
3rd November 2014 to 11th March 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
See test material information
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Samples were taken from the control and 100 mg/l loading rate WAF test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. Duplicate samples were taken at occasion and stored frozen for further analysis if necessary.
An additional sample of the 100 mg/l loading rate WAF test preparation was incubated alongside the test, remaining unopened until analysis at 72 hours.
- Sampling method: Not reported
- Sample storage conditions before analysis: The 0-h samples were frozen prior to analysis whilst the 72-h samples were analysed on the day of receipt.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A nominal amount of test item (560 mg) was added to the surface of 5.6 litres of culture medium in a sealed vessel with minimal headspace to give the 100 mg/L loading rate. 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 mixture allowed to stand for 1 hour. 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. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 100 mg/L loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present.

An aliquot (2 litres) of the WAF was inoculated with algal suspension (13.4 mL) to give the required test concentration of 100 mg/L loading rate WAF.

The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours.

Six flasks each completely filled with test preparation were used for the control and 100 mg/l loading rate WAF treatment group.

- Controls: The control group was maintained under identical conditions but not exposed to the test item.

- Evidence of undissolved material (e.g. precipitate, surface film, etc.): At the start and end of the mixing period, and following the 1 hour standing period, the 100 mg/l loading rate WAF was observed to have formed a clear colourless media column with oily globules of test item floating at the media surface. Microscopic evaluation of the WAF showed there to be no micro-dispertions of globules of test item present.

At the start of the test all control and test cultures were observed to be clear, colourless solutions.
After the 72-hour test period all control and test cultures were observed to be green dispersions.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Green algae
- Strain: CCAP 278/4
- Source (laboratory, culture collection): The Culture Collection of Algae and Protazoa (CCAP), SAMS Research Services Ltd., Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained at the laboratory by the periodic replenishment of culture medium.
- Age of inoculum (at test initiation): not reported
- Method of cultivation: Prior to the start of the test, sufficient master culture was added to approximately 100 ml volumes of culture media contained in conical flasks to give an initial cell density of approximately 10^3 cells/ml.

ACCLIMATION
- Acclimation period: The culture medium used for the tests was the same as that used to maintain the stock culture, with the addition of 500 mg/l of sodium bicarbonate to counteract the increase in pH due to algal growth in an enclosed system.
- Culturing media and conditions (same as test or not): Yes
- Any deformed or abnormal cells observed: None reported
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Hardness:
not reported
Test temperature:
24°C
pH:
Control: 8.3 - 9.5
100 mg/l WAF: 7.9 - 9.4
Dissolved oxygen:
not reported
Salinity:
freshwater
Conductivity:
not reported
Nominal and measured concentrations:
Nominal: 100 mg/l loading rate WAF
Measured: Spike recovery samples: nominal concentration 0.02 mg/l = measured concentration of test item 0.0152 mg/l.
nominal concentration 0.05 mg/l = measured concentration of test item 0.0499 mg/l.
nominal concentration 0.1 mg/l = measured concentration of test item 0.0891 mg/l.
Measurements of each individual constituent (decane, undecane, dodecane, tridecane) at 0 and 72 hours were all
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 ml glass conical flask
- Type (delete if not applicable): closed - sealed with a ground glass stopper
- Material, size, headspace, fill volume: Glass, 250 ml, no headspace, 250 ml fill volume
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): Static
- Renewal rate of test solution (frequency/flow rate): n/a
- Initial cells density: 5 x 10^3 cells/ml
- Control end cells density: 7.27 E+05
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): n/a

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used: n/a

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture with the addition of 500 mg/L of sodium bicarbonate to counteract the increase in pH due to algal growth in an enclosed system (Herman et al 1990). The culture medium was prepared using reverse osmosis purified deionised water and pH adjusted to 7.5 with 0.1N NaOH or HCL. For the purposes of the range-finding and definitive test, additional sodium bicarbonate (500 mg/l) was added to the prepared culture medium prior to use.
- Culture medium different from test medium: No
- Intervals of water quality measurement: pH of the control and 100 mg/l loading rate WAF was determined at initiation of the test and after 72 hours exposure. The temperature in the incubator was recorded daily.

OTHER TEST CONDITIONS
- Sterile test conditions: not reported
- Adjustment of pH: Yes
- Photoperiod: Continuous light
- Light intensity and quality: 7000 lux provided by warm white lighting (380-730 nm)
- Salinity (for marine algae): n/a
The flasks were sealed with ground glass stoppers and incubated at 24 (+/- 1) °C under continuous illumination and constantly shaken at approximately 150 rpm for 72 hours.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Cell densities were recorded at 0 and 72 hours in each replicate for the control and 100 mg/l loading rate WAF.
- Determination of cell concentrations: Coulter multisizer particle counter
- Chlorophyll measurement: no
- Other:

TEST CONCENTRATIONS
- Spacing factor for test concentrations: n/a
- Justification for using less concentrations than requested by guideline: No effects seen in range-finding test up to 100 mg/l WAF
- Range finding study
- Test concentrations: nominal loading rates of 10 and 100 mg/l WAF
- Results used to determine the conditions for the definitive study: After 72 hours the cell density of each test flask was determined using a Coulter Multisizer Particle Counter. No inhibition of growth rate was observed at the 10 or 100 mg/l loading rate WAF.
Reference substance (positive control):
yes
Remarks:
potassium dicromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF
Basis for effect:
other: yield
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF
Basis for effect:
other: yield
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): none reported
- Unusual cell shape: none reported
- Colour differences: none reported
- Flocculation: none reported
- Adherence to test vessels: none reported
- Aggregation of algal cells: none reported
- Other:
- Any stimulation of growth found in any treatment: Slight
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: n/a
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
- Results with reference substance valid: Yes
- ErC50: 1.2 mg/l
- Other: NOEC for growth rate = 0.5 mg/l

Table: Inhibition of growth rate and yield in the definitive test

 Nominal loading rate (mg/l)  Growth rate (cells/ml/hour) (mean of each replicate)    Yield (cells/ml)* (mean of each replicate)  
   0 -72 h  % inhibition  0 -72 h  % inhibition
 Control  0.069  -  7.22E+05  -
 100  0.071  [2]  8.16E+05  [13]

* In accordance with the OECD test guideline only the mean value for yield is calculated

[Increase in growth rate as compared to the controls]

Validity criteria fulfilled:
yes
Conclusions:
Measured toxicity data are available for Shell GTL Solvent GS190 (Hydrocarbons, C10-C13, n-alkanes, isoalkanes, <2% aromatics) to the freshwater green alga Pseudokirchneriella subcapitata. The test was conducted under static (no renewal of the test media) conditions in accordance with OECD Test Guideline 201 and method C3 of EC Regulation No. EC 761/2009. Appropriate modifications to the test and media preparation procedures were made to take account of the test substance containing multiple constituents, having low solubility in water and being potentially volatile. No effects on growth (expressed in terms of yield (y) and growth rate (r)) of P. subcapitata were observed after 72 hours exposure to the test medium prepared as a water-accommodated fraction (WAF) at a loading rate of 100 mg/l; 72-hour ErL50 value was >100 mg/l and NOELR value was 100 mg/l. The results of the test are considered to be reliable.
Executive summary:

Introduction

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 C.3 of Commission Regulation (EC) 761/2009.

 

Method

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

 

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to a Water Accommodated Fraction (WAF) of the test item, at a single nominal loading rate of 100 mg/L (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C.

 

Due to the potentially volatile nature of the test item, testing was conducted 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.

 

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

GC analysis of each individual constituent in the 100 mg/L loading rate WAF test preparations at 0 and 72 hours showed measured concentrations of less than the limit of quantification (LOQ).

 

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 Pseudokirchneriella subcapitata to the test item gave EL50 values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.

 

It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L loading rate WAF.

Description of key information

There is data available for this substance. Additionally, key and WOE data is available for structural analogues Hyd C10-C13, nic, <2% arom; Hyd C10-C13, ni, <2% arom. The data is read across to this substance based on analogue read across and a discussion and report on the read across strategy is provided as an attachment in IUCLID Section 13.

Growth of Pseudokirchneriella subcapitata cultures, as measured by biomass and growth rate, exposed to a water accommodated fraction of Hyd C10-C12, isoalkanes, <2% arom was not inhibited over 72 hours. The growth of algae in the the test systems was greater than the controls, which performed normally. Therefore, the 72-hr EL50 values for the two endpoints are reported as >1000 mg/L

Growth of Pseudokirchneriella subcapitata cultures, as measured by biomass and growth rate, exposed to a water accommodated fraction of Hyd C10-C13, nic, <2% arom was not inhibited over 72 hours. The growth of algae in the the test systems was greater than the controls, which performed normally. Therefore, the 72-hr EL50 values for the two endpoints are reported as >1000 mg/L.

Exposure of Pseudokirchneriella subcapitata to Hyd C10-C13, ni, <2% arom gave EL50 values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.

Exposure of Pseudokirchneriella subcapitata to Hyd C10-C13, ni, <2% arom gave EL50 values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.

Key value for chemical safety assessment

Additional information