L-97-034-PB

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27th - 31st July 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Pentaerythritol mixed esters with 2-methyl butyric acid, n-pentanoic acid, n-heptanoic acid, 3,5,5-trimethylhexanoic acid, n-octanoic acid, n-decanoic acid

Cas Number:

141686-49-9

IUPAC Name:

Pentaerythritol mixed esters with 2-methyl butyric acid, n-pentanoic acid, n-heptanoic acid, 3,5,5-trimethylhexanoic acid, n-octanoic acid, n-decanoic acid

Constituent 2

Reference substance name:

Trimethylol propane mixed esters with 2-methyl butyric acid, n-pentanoic acid, n-heptanoic acid, 3,5,5-trimethylhexanoic acid, n-octanoic acid, n-decanoic acid

Cas Number:

141686-50-2

IUPAC Name:

Trimethylol propane mixed esters with 2-methyl butyric acid, n-pentanoic acid, n-heptanoic acid, 3,5,5-trimethylhexanoic acid, n-octanoic acid, n-decanoic acid

Test material form:

liquid

Details on test material:

Identification: Hatcol ® 1772
Appearance/physical state: Clear colorless liquid
Purity: 100% (no solvent present)
Composition: 70% pentaerythritol esters described by CAS# 141686-49-9; 30% trimethylolpropane esters described by CAS# 141686-50-2

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

PREPARATION OF STANDARDS
Standard solutions of test material were prepared in hexane at a nominal concentration of 1000 mg/I.

PREPARATION OF TEST SAMPLES
A volume of sample was extracted with three portions (3 x 50 ml) of dichloromethane and the extracts filtered through anhydrous sodium sulphate. The combined extracts were evaporated to dryness and the residue re-dissolved in hexane.

STABILITY TESTING
A range of pre-study test samples was prepared in bottles and analysed initially and then after storage at ambient temperature in light and dark conditions for approximately 96 hours (equivalent to the study exposure period). The samples were prepared by "spiking" known volumes of medium with test material. Pre-study WAFs were prepared and analysed initially and then after storage at ambient temperature in light and dark conditions for approximately 96 hours (equivalent to the study exposure period).

Test organisms

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), Institute of Freshwater Ecology, Ferry House, Ambleside, Cumbria. Cultures were maintained in the laboratory by the periodic replenishment of culture medium. The culture was maintained in the laboratory at a temperature of 21 ± 1°C under continuous illumination (intensity approximately 7000 lux) and constant aeration.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Test conditions

Test temperature:

24 ± 1°C

pH:

The pH values of the control cultures were observed to increase from pH 7.5 at 0 hours to pH 9.1 - 10.0 at 96 hours. The increase in pH observed after 96 hour is considered not to affect the integrity of the study, given that the validation criterion for control cell multiplication was exceeded.

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

96 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Key result

Duration:

96 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Any other information on results incl. tables

Range-finding study

The results showed no effect on growth at the test concentrations of 10 and 100 mg/l loading rate WAF. Preliminary work conducted for the validation of mixing period indicated that there was no significant increase in the amount of Total Organic Carbon (TOC) in the WAF by extending the mixing period for longer than 24 hours.Therefore a 24-hour mixing period with an hour settlement period was considered appropriate for the test. Based on this information a single test concentration of six replicates, of 100 mg/l loading rate WAF was selected for the definitive study. This experimental design conforms to a "limit test" to confirm that at the maximum test concentration given in the test guidelines no effect on growth was observed.

Definitive study

Growth data: From the data it is clear that neither the growth rate (r) or the biomass (b) of Pseudokirchneriella subcapitata (CCAP 276/20) were affected by the presence of the test material over the 96 -hour exposure period. The ELR₅₀ value with respect to biomass, E_bLR₅₀ (72 and 96 h), was determined by inspection of the area under the growth curve data after 72 and 96 hours. The EC₅₀ value with respect to the growth rate, E_bLR₅₀ (0 - 96 h), was determined by inspection of the growth rates for the period 0 - 96 hours.

Accordingly the following results were determined from the data:

E_bLR₅₀ (96 h) - >100 mg/l loading rate WAF

E_rC₅₀ (0 - 96 h) - 100 mg/l loading rate WAF

where E_bLR₅₀ is the test concentration that reduced biomass by 50% and E_rC₅₀ is the test concentration that reduced specific growth by 50%.

Statistical analysis of the area under the growth curve data was carried out for the control and 100 mg/l loading rate WAF test group using the Students t-test. 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 Concentration' (NOEC) is given as equal to 100 mg/l loading rate WAF. It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/l loading rate WAF. The following data show that the cell concentration of the control cultures increased by a factor of 65 at 72 hours and by a factor of 231 at 96 hours during the test in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours, and the EPA Guidelines that states the enhancement must be at least by a factor of 100 after 96 hours:

Mean cell density of control at 0 hours: 1.22 x 10⁴ cells per ml

Mean cell density of control at 72 hours: 7.96 x 10⁵ cells per ml

Mean cell density of control at 96 hours: 2.82 x 10⁶ cells per ml

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Pseudokirchneriella subcapitata to the test material gave EL50 values of greater than 100 mg/L loading rate WAF. Correspondingly, the No Observed Effect Concentration (NOEC) was equal to 100 mg/L. It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L loading rate WAF.

Executive summary:

Introduction

A study was performed to assess the effect of the test material on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in the OECD Guidelines for Testing of Chemicals (1984) No 201; US CFR Title 40, Part 797, Section 1050; US EPA Pesticide Assessment Guideline, Sub-division J,Section 122 -2 and US EPA Draft Ecological Effects Test Guidelines OPPTS 850.5400.

Procedure

Following a preliminary range-finding study, Pseudokirchneriella subcapitata was exposed to a Water Accommodated Fraction (WAF) at a single loading rate of 100 mg/l (six replicate flasks) for 96 hours, under constant illumination and shaking at a temperature of 24 ± 1°C. Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group using a Coulter® Multisizer II Particle Counter.

Results

Exposure of Pseudokirchneriella subcapitata to the test material gave EL50 values of greater than 100 mg/l loading rate WAF. Correspondingly, the No Observed Effect Concentration (NOEC) was equal to 100 mg/l. It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/l loading rate WAF.