Alkanes, C14-20

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

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:

7th August 2014 to 19th August 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.

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

Details on sampling:

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.

Vehicle:

no

Details on test solutions:

 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 100 mg/L loading rate WAF was inoculated with algal suspension (17 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 103 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 ºC until the algal cell density was approximately 104 – 105 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

Not stated in report

Test temperature:

24 +/- 1°C recorded daily

pH:

Measured using a Hach HQ30d Flexi handheld meter.

Dissolved oxygen:

Not stated in report

Salinity:

Not applicable as a freshwater study

Nominal and measured concentrations:

Range finding test: 10 and 100 mg/L loading rate
Definitive test: 100 mg/L loading rate

Details on test 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 5.85 x 10^5 cells per mL. Inoculation of 2 litres of test medium with 17 mL of this algal suspension gave an initial nominal cell density of 5 x 10^3 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.

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:

other: loading rate 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: loading rate WAF

Basis for effect:

growth rate

Details on results:

From the data given in Tables 2 and 4 (please refer to the attached documents), 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.

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:

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 a Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981). There were no statistically significant decreases 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%.

Statistical analysis of the yield data was carried out as per the above. There were no statistically significant decreases 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.

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 (see Table 2) was observed to increase from pH 8.6 at 0 hours to pH 10.5 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 (see Figure 1) 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 100 mg/L loading rate WAF 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:

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated and 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.

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 (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

Total Organic Carbon (TOC) analysis of the 100 mg/L loading rate WAF test preparation at 0 hours showed a measured concentration of 6.14 mg C/L was obtained. A decline in measured carbon concentration was observed at 72 hours to 3.04 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 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.

Reference 2

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:

7th August 2014 to 19th August 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of rleevant results.

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

Details on sampling:

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.

Vehicle:

no

Details on test solutions:

 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 100 mg/L loading rate WAF was inoculated with algal suspension (17 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 103 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 104 – 105 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

Not stated in report

Test temperature:

24 +/- 1°C recorded daily

pH:

Measured using a Hach HQ30d Flexi handheld meter.

Dissolved oxygen:

Not stated in report

Salinity:

Not applicable as a freshwater study

Nominal and measured concentrations:

Range finding test: 10 and 100 mg/L loading rate
Definitive test: 100 mg/L loading rate

Details on test 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 5.85 x 10^5 cells per mL. Inoculation of 2 litres of test medium with 17 mL of this algal suspension gave an initial nominal cell density of 5 x 10^3 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.

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:

other: loading rate 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: loading rate WAF

Basis for effect:

growth rate

Details on results:

From the data given in Tables 2 and 4 (please refer to the attached documents), 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.

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:

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 a Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981). There were no statistically significant decreases in growth rate (P0.05), between the control and100 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%.

Statistical analysis of the yield data was carried out as per the above. There were no statistically significant decreases 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.

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 (see Table 2) 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 unites after 72 hours and was therefore 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 100 mg/L loading rate WAF 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:

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated and 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.

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 (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

Total Organic Carbon (TOC) analysis of the 100 mg/L loading rate WAF test preparation at 0 hours showed a measured concentration of 4.93 mg C/L was obtained. A decline in measured carbon concentration was observed at 72 hours to 0.79 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 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.

Reference 3

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study complies with ISO 10253:2006 testing method. However, the control of the Water Accomodated Fraction preparation is not mentioned, especially which the compounds present in the fraction and its stability. Also, an important incoherence in the results are hardly discussed (significant effect at 30.2 and 99.1 mg/L WAF but the NOEC is claimed at 993 mg/L WAF). MSDS provided.

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:

ISO 10253 (Water quality - Marine Algal Growth Inhibition Test with Skeletonema costatum and Phaeodactylum tricornutum)

Version / remarks:

NS-EN ISO 10253, 1998

Deviations:

no

Principles of method if other than guideline:

Guideline study

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
No test surrogate material is tested

Analytical monitoring:

no

Details on sampling:

Not applicable (no analytical monitoring)

Vehicle:

no

Details on test solutions:

The solubility of the test substance was reported to be <1 mg/L in water. Water accomodated fractions (WAFs) were prepared individually by stirring the appropriate amounts of the test substance in growth medium. The solutions were stirred with a spin-bar for 20 hours. Samples for testing were taken from the mid of the water phase with a peristaltic pump. Test results relate to these water accomodated fractions.

Test organisms (species):

Skeletonema costatum

Details on test organisms:

TEST ORGANISM
- Common name: Skeletonema costatum
- Source (laboratory, culture collection): Oslo fiord (1962)
- Strain: NIVA-BAC1
- Method of cultivation: incubated ca. 20°C under continuous light (>50 µE/m-2/sec-1)

ACCLIMATION
- Culturing media and conditions: same as test

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No post-exposure observation

Hardness:

Not relevant (seawater study)

Test temperature:

20 ±2°C

pH:

8.2-8.3

Dissolved oxygen:

No data provided

Salinity:

33.9 per thousand

Nominal and measured concentrations:

2.4, 9.7, 32.2, 99.1, 320, 993 and 3200 mg/l (nominal loading rates).

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: 100 mL flat-bottomed flasks on an orbital shaker
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: the seawater was used as basis for the growth medium. The water was filtered through a GF/C filter, and briefly heated to about 75°C prior to use.

OTHER TEST CONDITIONS
- Sterile test conditions: No
- Adjustment of pH: No
- Photoperiod: continuous light
- Light intensity and quality: >50 µE/m-2/sec-1
- Salinity (for marine algae): 33.9 per thousand

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Algal growth measured by fluorescence: Turner Designs, Model 10 fluorimeter


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.33

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol 97%

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 3 200 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: (based upon nominal loading rates)

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

993 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: (based upon nominal loading rates)

Details on results:

- Exponential growth in the control (for algal test): yes
- Any stimulation of growth found in any treatment: the 72 h NOELR was 993 mg/L but a significant inhibition of growth rate was observed at 32.2 and 99.1 mg/L (nominal loading rates)

Results with reference substance (positive control):

Test concentration: 3 x 1.5 mg/L
Inhibition (mean value): 1.04 - 1.25 - 1.40 (growth rate)
- pH: at start 8.3 - at end 8.2

Reported statistics and error estimates:

Not relevant

During the test, the fluorescence (growth measurements) was recorded after 24, 48 and 72 +/- 2 hours and the pH was recorded at start and at end of test.

Validity criteria fulfilled:

yes

Conclusions:

The 72 hour EL50 was >3200 mg/L (based upon loading rates).

Executive summary:

The solubility of the test substance was reported to be < 1 mg/L in water. WAF were prepared by stirring the product in growth medium for 20 hours, followed by standstill for 4 hours. Samples for testing were taken from the mid of the water phase. Test cultures of 50 mL were incubated in 100 mL flat-bottomed flasks on an orbital shaker, 3 replicates for each concentration. Test substance concentrations from 2.4 mg/L to 3200 mg/L (based upon nominal loading rates) were inoculated to about 2000 cells/mL from an exponentially growing inoculum culture.

The test was run for about 72 hours under continuous light from fluorescent tubes of universal natural white type, at a temperature of 18.9 - 20.1°C. Six flat-bottomed flasks contained blank and three flasks contained the reference substance 3.5 -Dichlorophenol at 1.5 mg/L. Sub-samples of 5 mL were taken from the test flasks each day for growth measurements.

The inhibitory effect on the growth of Skeletonema costatum is expressed as the concentration of a product giving 50% of growth rate (ErC₅₀). The programme also calculates concentrations inhibiting growth rate 10% and 90% (EC₁₀ and EC₉₀). However, the results are based on nominal loadings. Therefore they are expressed here as EL (Effective Loading), instead of EC (Effective Concentration) as in the original report study.

The 72 hour EL₅₀ with DEV 2006 -16 was > 3200 mg/L (based on nominal laoding rates).

Calculation of NOELR is done by single-factor ANOVA followed by Dunnetts procedure for comparing each concentration mean with the control mean.

The 72 h NOELR was 993 mg/L but a significant inhibition of growth rate was observed at 32.2 and 99.1 mg/L (based on nominal loading rates).

All the criteria for test acceptance were fulfilled.

72h-EL₅₀ > 3200 mg/L (based upon nominal loading rates)

72h-NOELR = 993 mg/L (based upon nominal loading rates)

Reference 4

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: No inhibitory effect even with the highest concentration. EL50 not reached. Based on WAFs.

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

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

ISO 10253 (Water quality - Marine Algal Growth Inhibition Test with Skeletonema costatum and Phaeodactylum tricornutum)

Deviations:

no

Principles of method if other than guideline:

Guideline study

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
No test surrogate material is tested

Analytical monitoring:

no

Details on sampling:

Not applicable (no analytical monitoring)

Vehicle:

no

Details on test solutions:

The test solutions were prepared as water accomodated fractions (WAFs). They were prepared by direct addition of the required nominal amounts, followed by stirrring for approximately 20 hours. The preparations were then allowed to stand for appromately 1 h, before withdrawing a sample by siphon from the middle of the water phase, avoiding incorporation of floating/settled material.

Test organisms (species):

Skeletonema costatum

Details on test organisms:

TEST ORGANISM
- Common name: Skeletonema costatum
- Age of inoculum (at test initiation): at least 4 days
- Method of cultivation: Stock culture in laboratory at ca. 20°C at constant illumination

ACCLIMATION
- Acclimation period: 4 days
- Culturing media and conditions: same as test

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No post exposure observation

Hardness:

Not relevant (seawater study)

Test temperature:

19.5 °C

pH:

8.07 - 8.81

Dissolved oxygen:

No data provided

Salinity:

No data provided

Nominal and measured concentrations:

10096, 20063, 30025, 60085 and 100204 mg/L (ppm). These concentrations are based after weighing the test substance in the algal culture medium.

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type: closed (all flasks looselys covered with aluminium foil caps)
- Material, size, headspace, fill volume: borosilicate glass conical flasks, 80 mL test medium
- Aeration: Not provided in the study report
- Initial cells density:10^4 cells/ml
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 4

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: No
- Adjustment of pH: Yes
- Photoperiod: continuous light
- Light intensity and quality: 4 x 40W tubes mounted at a distance of 40 cm directely above the test area

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: counting chamber

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8

Reference substance (positive control):

yes

Remarks:

Water-soluble chemical 3',5-dichlorophenol

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 10 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Not relevant

Details on results:

- Exponential growth in the control (for algal test): yes

Results with reference substance (positive control):

72 h LC50 = 3.83 mg/L

Reported statistics and error estimates:

Not relevant as the EL50 is greater than the highest tested concentration.

No remarks

Validity criteria fulfilled:

yes

Conclusions:

No growth inhibition at concentrations up to 10,000 mg/L

Executive summary:

The test procedure followed is described in the ISO/DIS 10253 « Water quality » - Marine algal growth inhibition test with Skeletonema costatum and Phaeodactylum tricornutum”, 1991.

Skeletonema costatum was used as test organism.

The organisms are exposed to Water Accomodated Fractions (WAFs) of test substance H383 -96.

The exposure of Skeletonema costatum to concentrations up to 10,000 mg/L for 72 h does not inhibit algal growth (based on growth rate measurements).

Reference 5

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:

March to November 1993

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:

yes

Details on sampling:

An equilibration study was conducted prior to the study to determine the length of time needed to develop water accommodated fractions (WAFs) at the 1000 mg/L loading level. The aqueous phase of the WAF was analyzed after 1, 24, 48, and 96 hours of stirring. In all samples, the quantity of test substance in solution was less than the limit of detection (0.5 mg/L). Further analysis of test substance in test media a loading level of 1 g/L also showed no detectable amounts of test substance. Consequently, analysis of treatment solutions from the definitive study were not conducted.

Vehicle:

no

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

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

None

Test temperature:

23 to 25 degrees C

pH:

7.2 to 8.8

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 that were complely filled with test media. Six replicate test systems were established for the treatment level and control. 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

Validity criteria fulfilled:

yes

Conclusions:

Growth of an alga culture, Pseudokirchnerella subcapitata, as measured by biomass and growth rate, exposed to a 1000 mg/L water accommodated fraction of the test substance was not inhibited when compared to the control after a 72-hour exposure. Therefore, the 72-hr EL50 and NOELR values for these two endpoints are reported as >1000 mg/L and 1000 mg/L, respectively.

Executive summary:

Growth of an alga culture, Pseudokirchnerella subcapitata, as measured by biomass and growth rate, exposed to a 1000 mg/L water accommodated fraction of the test substance was not inhibited when compared to the control after a 72-hour exposure. Therefore, the 72-hr EL50 and NOELR values for these two endpoints are reported as >1000 mg/L and 1000 mg/L, respectively.

Description of key information

There is no data available for this substance. However, key and supporting data is available for structural analogues. 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. Key information is summarised below:

Hydrocarbons, C14-C16, n-alkanes, isoalkanes, <2% aromatics presented a 72-h EL50 (growth rate) for Pseudokirchneriella subcapitata of >100 mg/L (WAF) and a 72-h NOELR (growth rate) for Pseudokirchneriella subcapitata of 100 mg/L.

Hydrocarbons, C15-C19, n-alkanes, isoalkanes, <2% aromatics presented a 72-h EL50 (growth rate) for Pseudokirchneriella subcapitata of >100 mg/L (WAF) and a 72-h NOELR (growth rate) for Pseudokirchneriella subcapitata of 100 mg/L.

Hydrocarbons, C14-C18, n-alkanes, isoalkanes, cyclics, <2% aromatics presented a 72-h EL50 (growth rate) for Skeletonema costatum of >3200 mg/L (based upon loading rates) and a 72-h NOELR (growth rate) for Skeletonema costatum of 993 mg/L.

Hydrocarbons, C14-C18, n-alkanes, isoalkanes, cyclics, <2% aromatics presented a 72-h EL50 (growth rate) for Skeletonema costatum of >10000 mg/L.

Hydrocarbons, C11-C14, n-alkanes, isoalkanes, cyclics, <2% aromatics presented a 72-h EL50 (growth rate) for Pseudokirchnerella subcapitata of >1000 mg/L and a 72-h NOELR (growth rate) for Pseudokirchnerella subcapitata of 1000 mg/L.

The toxicity level was never reached in any study despite the high loading level tested. Therefore, this substance is considered to display no acute toxicity to algae based on an analogue read across approach.

Additional information

The substances are poorly soluble in water, most of the studies are performed using Water accommodated fractions (WAFs) and all the results are based on nominal loadings. Thus, the values indicated below as NOECs are actually NOELRs (No observed effect loading rates). No study could lead to the calculation of EL₅₀.

 

Seventeen study reports were available for this end-point. Fourteen delt with the toxicity of the substance Hydrocarbons, C14-C18, n-alkanes, isoalkanes, cyclics, < 2% aromatics on Skeletonema costatum growth rate, while three other substances (Hydrocarbons, C14-C16, n-alkanes, isoalkanes, <2% aromatics; Hydrocarbons, C15-C19, n-alkanes, isoalkanes, <2% aromatics; and Hydrocarbons, C11-C14, n-alkanes, isoalkanes, cyclics, <2% aromatics) dealt with the toxicity on Pseudokirchnerella subcapitata growth rate.

None of the studies using the marine species Skeletonema costatum on Hydrocarbons, C14-C18, n-alkanes, isoalkanes, cyclics, < 2% aromatics allowed calculating 48h-EL₅₀that was never reached. All EL₅₀ are superior to 1,000 mg/L based on WAF studies.

Growth of Pseudokirchnerella subcapitata culture exposed to a 100 mg/L water accommodated fraction (for Hydrocarbons, C14-C16, n-alkanes, isoalkanes, <2% aromatics; and Hydrocarbons, C15-C19, n-alkanes, isoalkanes, <2% aromatics) or 1000 mg/L water accommodated fraction (for Hydrocarbons, C11-C14, n-alkanes, isoalkanes, cyclics, <2% aromatics) was not inhibited when compared to the control after a 72-hour exposure. Therefore, the 72-hr EL₅₀ values are reported as >100 mg/L and >1000 mg/L, respectively, and the NOELR values are reported as 100 mg/L and 1000 mg/L, respectively.