Soybean oil, maleated, ester with triethanolamine

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date 01 May 2017 Experimental completion date 26 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

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.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Physical state/Appearance: Clear amber viscous liquid

Purity: 100% product
Expiry Date: 24 June 2018
Storage Conditions: Room temperature in the dark

Analytical monitoring:

yes

Remarks:

Analysis of the WAFs was carried out by Total Organic Carbon (TOC) analysis. Samples were taken from the uninoculated control and each loading rate WAF test group at 0 and 72 hours for this analysis

Vehicle:

no

Details on test solutions:

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. A study to determine the General Physico-Chemical Properties of the Test Item (Envigo Study Number YK58VV) indicated the water solubility of the test item was 6.89 x 10-2 g C/L. Given this it was considered appropriate to test up to a maximum loading rate of 100 mg/L. A 23-Hour stirring period, followed by a 1-Hour standing period was deemed sufficient to ensure equilibration between the test item and aqueous phase.

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 ± 2 Deg 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 Deg C until the algal cell density was approximately 104 – 105 cells/mL.
A positive control (Envigo Study Number FP48BQ) used potassium dichromate as the reference item. Details of the positive control are given in the attached Annex 2. The positive control was conducted between 28 November 2016 and 01 December 2016.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

Not reported

Test temperature:

24 ± 1 °C

pH:

7.4 to 7.6

Dissolved oxygen:

Not reported

Salinity:

N/A

Conductivity:

N/A

Nominal and measured concentrations:

Based on the results of the range-finding test, and following discussions with the Sponsor, the following loading rates were assigned to the definitive test: 1.25, 4.0, 12.5, 40 and 125 mg/L.

Reference substance (positive control):

yes

Remarks:

A positive control (Envigo Study Number FP48BQ) used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L.

Key result

Duration:

72 h

Dose descriptor:

EL50

Remarks:

(mg/L Loading Rate WAF)

Effect conc.:

60 mg/L

Nominal / measured:

nominal

Conc. based on:

other: (mg/L Loading Rate WAF)

Basis for effect:

growth rate

Remarks on result:

other: EL50 (mg/L Loading Rate WAF)

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

33 mg/L

Nominal / measured:

nominal

Conc. based on:

other: (mg/L Loading Rate WAF)

Basis for effect:

other: Yield

Remarks on result:

other: EL50 (mg/L Loading Rate WAF)

Key result

Duration:

72 h

Dose descriptor:

other: NOEL

Effect conc.:

12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

other: No Observed Effect Loading Rate (NOEL) (mg/L)

Basis for effect:

growth rate

Remarks on result:

other: No Observed Effect Loading Rate (NOEL) (mg/L)

Key result

Duration:

72 h

Dose descriptor:

other: NOEL

Effect conc.:

12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

other: No Observed Effect Loading Rate (NOEL) (mg/L)

Basis for effect:

other: Yield

Remarks on result:

other: No Observed Effect Loading Rate (NOEL) (mg/L)

Key result

Duration:

72 h

Dose descriptor:

other: LOEL

Effect conc.:

40 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Lowest Observed Effect Loading Rate (LOEL) (mg/L)

Basis for effect:

growth rate

Remarks on result:

other: Lowest Observed Effect Loading Rate (LOEL) (mg/L)

Key result

Duration:

72 h

Dose descriptor:

other: LOEL

Effect conc.:

40 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Lowest Observed Effect Loading Rate (LOEL) (mg/L)

Basis for effect:

other: Yield

Remarks on result:

other: Lowest Observed Effect Loading Rate (LOEL) (mg/L)

Results with reference substance (positive control):

A positive control (Envigo Study Number FP48BQ) 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 – 72h)          :          1.4 mg/L; 95% confidence limits 1.2 – 1.5mg/L

EyC50(0 – 72 h)         :          0.60 mg/L; 95% confidence limits 0.52 – 0.69mg/L

No Observed Effect Concentration (NOEC) based on growth rate:                   0.25mg/L
No Observed Effect Concentration (NOEC) based on yield:                             0.25mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate:            0.50 mg/L
Lowest Observed Effect Concentration (LOEC) based on yield:                        0.50mg/L
The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Range-finding Test

 

The cell densities and percentage inhibition of growth values from the exposure of Pseudokirchneriella subcapitata to the test item during the range-finding test are given in Table 1.

 

The results showed no significant effect on growth rate at 10 mg/L loading rate WAF. However, growth was observed to be reduced at 100 mg/L loading rate WAF.

 

Based on this information loading rates of 1.25, 4.0, 12.5, 40 and 125 mg/L were selected for the definitive test.

 

Definitive Test

Total Organic Carbon Analysis

 

Total Organic Carbon (TOC) analysis of the WAFs containing no algal cells conducted at 0 and 72 hours (see Annex 4) showed measured concentrations to range from 1.6 to 53 mg C/L.

 

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

 

Growth Data

 

Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 2. Daily specific growth rates for the control cultures are given in Table 3. Growth rate and yield values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 4.

 

The mean cell densities versus time for the definitive test are presented in Figure 1. Percentage inhibition values are plotted against loading rate in Figure 2 and Figure 3.

 

From the data given in Tables 2 and 4, it is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were affected by the presence of the test item over the 72-Hour exposure period.

 

Accordingly the following results were determined from the data:

 

Inhibition of growth rate

 

ErL10 (0 - 72 h) : 14 mg/L loading rate WAF

ErL20 (0 - 72 h) : 24 mg/L loading rate WAF

ErL50 (0 - 72 h)   :   60 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 all loading rates using one way analysis of variance 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 between the control, 1.25, 4.0 and 12.5 mg/L loading rate WAFs (P0.05), however all other loading rates were significantly different (P<0.05) and, therefore the "No Observed Effect Loading Rate" (NOEL) based on growth rate was 12.5 mg/L loading rate WAF. Correspondingly the "Lowest Observed Effect Loading Rate" (LOEL) based on growth rate was 40 mg/L loading rate WAF.

 

Inhibition of Yield

 

EyL10 (0 - 72 h) : 26 mg/L loading rate WAF

EyL20 (0 - 72 h) : 29 mg/L loading rate WAF

EyL50 (0 - 72 h)  :   33 mg/L loading rate WAF

 

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

Statistical analysis of the yield data was carried out as in Section 6.2.3. There were no statistically significant differences between the control, 1.25, 4.0 and 12.5 mg/L loading rate WAFs (P0.05), however all other loading rates were significantly different (P<0.05) and, therefore the "No Observed Effect Loading Rate" (NOEL) based on yield was 12.5 mg/L loading rate WAF. Correspondingly the "Lowest Observed Effect Loading Rate" (LOEL) based on yield was 40 mg/L loading rate WAF.

 

 

Validation Criteria

 

The following data show that the cell concentration of the control cultures increased by a factor of 133 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.

Nominal cell density of control at 0 hours             :              5.00 x 103 cells per mL Mean cell density of control at 72 hours   :              6.67 x 105 cells per mL

 

The mean coefficient of variation for section by section specific growth rate for the control cultures was 29% 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 1% 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

 

The pH values of the control and each test concentration are given in Table 2. Temperature was maintained at 24 ± 1 ºC throughout the test.

 

 

 

The pH value of the control cultures (see Table 2) was determined to be pH 7.4 at both 0 and 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 WAFs.

 

At the start of mixing the 1.25 mg/L loading rate WAF was observed to have formed a clear colorless media column with test item floating at the media surface. The 4.0 and 12.5 mg/L loading rate WAFs were clear colorless media columns with test item floating at the media surface and throughout the media column whilst the 40 and 125 mg/L loading rate WAFs formed hazy dispersions instantly. After stirring, and following a 1-Hour standing period both the 1.25 and 4.0 mg/L loading rate WAFs were observed to have formed clear colorless media columns with test item floating at the media surface and suspended within the media column. The 12.5, 40 and 125 mg/L loading rate WAFs were observed to have formed hazy non-homogenous dispersions, the opacity of which increased with increasing loading rate.

As visual observations made indicated that undissolved test item was present it was considered appropriate to remove the aqueous phase by filtration through a glass wool plug and one sheet of Postlip filter paper. Following filtration, no micro-dispersions of test item were observed to be present in the either the 1.25 or 4.0 mg/L loading rate WAFs. Dispersed test item remained visible in the 12.5, 40 and 125 mg/L loading rate WAFs however further filtration was considered unlikely to be able to remove any more undissolved test item.

 

At the start of the test all control, 1.25 and 4.0 mg/L loading rate WAF test cultures were observed to be clear colorless solutions. The 12.5 mg/L loading rate WAF cultures were extremely slightly hazy dispersions, the 40 mg/L loading rate WAF cultures were hazy dispersions and the 125 mg/L loading rate WAF cultures were very hazy dispersions. After the 72-Hour test period all control, 1.25, 4.0 and 12.5 mg/L loading rate WAF test cultures were observed to be green dispersions. The 40 mg/L loading rate WAF test cultures were hazy dispersions whilst the 125 mg/L loading rate WAF test cultures were very hazy dispersions.

Validity criteria fulfilled:

yes

Conclusions:

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

Response Variable EL50 No Observed Effect Lowest Observed Effect
(mg/L Loading Rate WAF) L oading Rate (NOEL) (mg/L) Loading Rate (LOEL) (mg/L)
Growth Rate 60 12.5 40
Yield 33 12.5 40

Executive summary:

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

 

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 Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 1.25, 4.0, 12.5, 40 and 125 mg/L (three replicate flasks per concentration) for

72 hours, under constant illumination and shaking at a temperature of 24 ± 1 Deg C.

 

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a haemocytometer and light microscope.

 

Total Organic Carbon (TOC) analysis of the test preparations was performed at 0 and 72 hours and showed measured concentrations to range from 1.6 to 53 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 the following results:

Response Variable	EL50 (mg/L Loading Rate WAF)	No Observed Effect Loading Rate (NOEL) (mg/L)	Lowest Observed Effect Loading Rate (LOEL) (mg/L)
Growth Rate	60	12.5	40
Yield	33	12.5	40

Description of key information

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

Response Variable	EL50 (mg/L Loading Rate WAF)	No Observed Effect Loading Rate (NOEL) (mg/L)	Lowest Observed Effect Loading Rate (LOEL) (mg/L)
Growth Rate	60	12.5	40
Yield	33	12.5	40

Key value for chemical safety assessment

EC50 for freshwater algae:

60 mg/L

EC10 or NOEC for freshwater algae:

12.5 mg/L

Additional information

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.

 

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 Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 1.25, 4.0, 12.5, 40 and 125 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 Deg C.

 

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a haemocytometer and light microscope.

 

Total Organic Carbon (TOC) analysis of the test preparations was performed at 0 and 72 hours and showed measured concentrations to range from 1.6 to 53 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 the following results:

Response Variable	EL50 (mg/L Loading Rate WAF)	No Observed Effect Loading Rate (NOEL) (mg/L)	Lowest Observed Effect Loading Rate (LOEL) (mg/L)
Growth Rate	60	12.5	40
Yield	33	12.5	40