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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:
The study was conducted between 07 August 2014 and 19 August 2014.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.
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
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis 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:
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 liters 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 liter) of each of the loading rate WAFs was separately inoculated with algal suspension (5.6 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 liters 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 liters) of the WAF was inoculated with algal suspension (13.6 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. Data from the control group was shared with similar concurrent studies.

Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 7.34E+05 cells per mL. Inoculation of 2 liters of test medium with 13.6 mL of this algal suspension gave an initial nominal cell density of 5 x 103 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
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: The toxicity cannot be attributed to a single constituent or a mixture of constituents but to the test item as a whole.
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
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 preparation at 0 hours showed a measured concentration of 1.82 mg C/L was obtained. A decline in measured carbon concentration was observed at 72 hours to 0.98 mg C/L.

The TOC in the control and 100 mg/L loading rate WAF at 0 hours was slightly higher than would normally be expected. Examination of the data could not find a reason for these high values, however as there was no inhibition of growth this was considered not to have affected the outcome or integrity of the study.


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 in growth rate (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 in yield 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 58 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: 7.34 x 103cells per mL

Mean cell density of control at 72 hours: 4.26 x 105cells per mL

 

The mean coefficient of variation for section by section specific growth rate for the control cultures was 20% 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 8.7 at 0 hours to pH 9.6 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.

 

 

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:
to the freshwater green alga Pseudokirchneriella subcapitata. The test was conducted under static conditions (no renewal of the test media) in accordance with OECD Test Guideline 201. 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 of P. subcapitata (expressed in terms of average specific growth rate (µ) and the area under the growth curve (A)) were determined after 72 hours of incubation in the test medium prepared as a water-accommodated fraction (WAF) at a loading rate of 100 mg/l; the EL50 values for both growth parameters were >100 mg/l and the NOELRs were >100 mg/l. Total Organic Carbon (TOC) analysis of the 100 mg/l loading rate WAF test preparation at 0 hours showed a measured concentration of 1.82 mg C/l. A decline in measured TOC was observed at 72 hours to 0.98 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 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 (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 preparation at 0 hours showed a measured concentration of 1.82 mg C/L was obtained. A decline in measured carbon concentration was observed at 72 hours to 0.98 mg C/L.

 

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.

Description of key information

72-h) ErL50 for algae (Pseudokirchneriella subcapitata): >100 mg/l [OECD 201; test mat. (WAFs) (nominal) based on: growth rate];

(72 h) NOELR for algae (Pseudokirchneriella subcapitata): ≥100 mg/l [OECD 201; test mat. (WAFs) (nominal) based on: growth rate].

Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L
EC10 or NOEC for freshwater algae:
100 mg/L

Additional information

Hydrocarbons, C18-C24, isoalkanes, <2% aromatics

Measured toxicity data are available for Hydrocarbons, C18-C24, isoalkanes, <2% aromatics to the freshwater green alga Pseudokirchneriella subcapitata (Vryenhoef, 2014b). The test was conducted under static conditions (no renewal of the test media) in accordance with OECD Test Guideline 201. 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 of P. subcapitata (expressed in terms of average specific growth rate (µ) and the area under the growth curve (A)) were determined after 72 hours of incubation in the test medium prepared as a water-accommodated fraction (WAF) at a loading rate of 100 mg/l; the EL50 values for both growth parameters were >100 mg/l and the NOELRs were >100 mg/l. Total Organic Carbon (TOC) analysis of the 100 mg/l loading rate WAF test preparation at 0 hours showed a measured concentration of 1.82 mg C/l. A decline in measured TOC was observed at 72 hours to 0.98 mg C/l. The results of the test are considered to be reliable.

GTL Gasoil

The toxicity of a sample of GTL Gasoil has been determined by Palmer (2001) in a test with the unicellular alga Raphidocelis subcapitata. The tests were conducted in accordance with OECD Test Guideline 201.

WAFs of the sample were prepared in sealed vessels with minimum headspace by stirring for approximately 72 hours. The contents of the vessels were left to stand for 1-2 hours before drawing off the aqueous phase – the WAF – for testing. Static exposures were then carried out in completely full, sealed vessels. The tests were not subject to GLP and the test media were not analysed for stability or for exposure concentration of the test substance. However in other respects they were considered to fulfil the requirements of current best practice.

The test results, expressed as the EL50 and NOEL values, showed that the sample was not acutely toxic to algae at a loading rate of 1000 mg/l.

Albertus and Phillips (2005) have reported results for an acute toxicity test carried out on a second sample of GTL Gasoil with the freshwater alga Selenastrum capricornutum.

The test was carried out using a scaled-down version of the US EPA algal bottle test method. In all cases the test media were water-accommodated fractions of the test sample that were prepared in sealed vessels with a small headspace according to the CONCAWE methodology for the ecotoxicological testing of petroleum products (CONCAWE report, 1993).

The test results, expressed as the EL50 and NOEL values, showed that the sample was not acutely toxic to algae at a loading rate of 1000 mg/l.

An algal growth and inhibition study OECD TG 201 (2011) was conducted for GTL Gasoil using Pseudokirchneriella subcapitata, (Chen, 2015).  A WAF loading rate of 100 mg/L was used to assess potential inhibitory effects to algal biomass after 24, 48, and 72-hour exposure. Once the test solutions were inoculated with the algae, the flasks were incubated at 22.5°C - 23.4°C and maintained under continuous fluorescent illumination (5990 - 6080 lux) for the duration of the study. The DOC concentrations of each test group were measured at 0, 24, 48, and 72-hours.  

The average specific growth rate of alga exposed to WAF loading rate of 100 mg/L was not significantly different (P ≤0.05) to that of the control at any of the time points in the study.  Therefore, the inhibition growth rate (72-hour ErL50) was determined to be ≥100 mg /L, based on nominal loading rate WAF. This study complied with the Chinese State Environmental Protection Administration (SEPA 2004) requirements rather than GLP but is considered reliable without restrictions and given a reliability score corresponding to Klimisch 1.

Discussion on methodology

In undertaking chronic toxicity testing the best use of all available data should be made. However, the assessment of ecotoxicity data for many petroleum products is complicated as products are UVBCs containing many individual constituents with a range of solubilities. Chemical analyses of the aqueous concentrations of all constituents are not possible due to the complexity of the composition.

The concentration of each constituent dissolved in the water phase at any particular ‘loading’ should be maximized. The maximum possible water concentration of each constituent is typically achieved through prolonged stirring of the water-petroleum substance mixture to produce a Water Accommodated Fraction (WAF). WAFs are prepared individually and not by serial dilution of a single stock solution.  In addition, a sealed system approach may be appropriate for more volatile petroleum substances.

GTL Gasoil is a poorly water soluble UVBC and poses specific challenges when preparing aqueous solutions for toxicity testing.  This substance contains constituents with a range of physio-chemical properties (e.g. volatility, water solubility) and fall under the OECD guidance document 23 (2nd Edition) description and definition of “difficult to test” substances (Organisation for Economic Co-operation and Development [OECD] 2019). All long-term aquatic studies with GTL Gas oil followed the recommendations laid out for such substances.

GTL Base oil distillates

The toxicity of a sample of GTL Base Oil Distillates has been determined by Harlan (Vryenhoef, 2009) in a test with the unicellular alga Desmodesmus subspicatus. The tests were conducted in accordance with OECD Test Guideline 201.

Desmodesmus subspicatus were exposed to water accommodated fractions (WAFs) of the test material over a single nominal loading rates of 100 mg/l for a period of 72 hours. The WAFs were prepared by stirring for 23 hour and left to stand for 1 hour before drawing off the aqueous phase – the WAF – for testing. Static exposures were then carried. The study was carried out according to GLP and analytical monitoring, TOC analysis, also took place. 

The test results, expressed as the EL50and NOELR values, showed that the sample was not toxic to algae at a loading rate of 100 mg/l.

Total Organic Carbon (TOC) analysis of the freshly prepared test preparations showed amount of carbon present within the 100 mg/l loading rate WAF test vessels to be less than the limit of quantitation (LOQ) for the method (1.0 mg C/l) in fresh and old test media samples.

Conclusion

Algal growth inhibition tests are available for the registered substance and for two GTL-derived substances in the relevant carbon number range for the registered substance. In the absence of adverse effects in these studies it can be concluded that, based on weight of evidence, the ELR50(algae) for Hydrocarbons, C18 -C24, isoalkanes, <2% aromatics is >100 mg/l and the NOELR is 100 mg/l.