1,4-dioxane-2,5-dione

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:

2001-11-16 to 2002-02-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

1984

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Glycollic acid

EC Number:

201-180-5

EC Name:

Glycollic acid

Cas Number:

79-14-1

Molecular formula:

C2H4O3

IUPAC Name:

glycol acid

Test material form:

liquid

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 10, 20, 40, and 75 mg/L
- Sampling method: Samples from each of the working stock, test concentrations, and blank control solutions were collected on day 0. An aliquot from each of the test concentrations and blank control solutions was taken prior to the addition of Selenastrum capricornutum. Samples from each of the test concentrations, blank control, and abiotic control solutions also
were collected on day 3. These samples were prepared by pooling all 3 replicates for each of the test and control solutions. Aliquots were taken from each of the pooled samples and given to the analytical chemist for analysis.
- Sample storage conditions before analysis: Samples were analysed on the day of receipt.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Preparation of AAP nutient medium: Adding 1mL of each of 6 macronutrient stock solutions and 1 mL of the micronutrient stock solution to approximately 800 mL of Milli-Q water (deionized), with mixing after addition. The volume was brought to 1 L with additional Milli-Q water. The test stock solution consisted of nominal 100 mg/L glycolic acid in 1000 mL sterilized AAP nutrient medium. The other nominal concentrations used in the test were made by respective dilutions of this stock solution.

Test organisms

Test organisms (species):

Selenastrum sp.

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: Selenastrum sp.
- Strain: Selenastrum capricornutum
- Method of cultivation: The organisms were cultured in sterilized 250 mL Erlenmeyer flasks containing approximately 50 mL of filtered (= filter-sterilized) synthetic algal-assay-procedure (AAP) nutrient medium and were aseptically transferred to fresh medium every 3 to 7 days. The flasks were fitted with foam stoppers to permit gas exchange.

ACCLIMATION
- Culturing media and conditions (same as test or not): same as test

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

At the end of the 72-hour exposure period, a single randomly selected replicate from the blank control and an aliquot from each of the replicate flasks from each of the test concentrations exhibiting a 50% or greater inhibition of cell counts relative to the blank control (nominal 40, 75, and 100 mg/L) were selected for the recovery test.

Test conditions

Test temperature:

24 ± 2°C

pH:

pH at test start:
concentration:
10 mg/L 7.39
20 mg/L 6.74
40 mg/L 5.16
75 mg/L 3.96
100 mg/L 3.70

pH at the end of exposure
10 mg/L 7.63
20 mg/L 7.55
40 mg/L 7.37
75 mg/L 7.39
100 mg/L 7.29

Nominal and measured concentrations:

Nominal concentrations: 0, 10, 20, 40, 75 and 100 mg/L
Measured concentrations: 0, 7.52, 14.5, 30.3 and 54.6 mg/L, which represents 106, 102, 107 and 103% recovery of the active ingredient.

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: Glass, 250 mL fill volume, 50 mL solution volume
- Initial cells density: 10,000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-Q water
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: Illumination: with 6000 to 10000 lux for 24h

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The counts were conducted using a hemacytometer and a compound microscope. An aliquot of each sample was loaded into the hemacytometer and 16 grids were selected. All cells located in the 16 grids were counted and recorded as healthy or unhealthy.

TEST CONCENTRATIONS
- Test concentrations: 10, 20, 40, 75 and 100 mg/L

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

44 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

20 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

22.5 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

cell number

Effect concentrations 4

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

cell number

Reported statistics and error estimates:

The mean healthy cell count, area under the growth curve, and growth rate at the 72-hour interval for each test concentration were expressed relative to the blank control. The growth rate and the percent inhibition as well as the EC50 value and the NOEC were calculated as described previously (OECD Test Guideline 201).

Any other information on results incl. tables

Table 1: Effect of glycolic acid on growth of Selenastrum capricornutum

Treatment nominal concentration (mg/l)		Initial cell density	Cell density at
			24 hours	48 hours	72hours
					Cell count	% inhibition (mean)
Negative control	Replicate #1	10,000	100,000	260,000	3,900,000
	Replicate #2	10,000	150,000	720,000	3,920,000
	Replicate #3	10,000	70,000	580,000	3,630,000
10	Replicate #1	10,000	70,000	320,000	3,710,000	2.27
	Replicate #2	10,000	140,000	290,000	3,630,000
	Replicate #3	10,000	110,000	460,000	3,850,000
20	Replicate #1	10,000	20,000	270,000	2,370,000	37.21
	Replicate #2	10,000	20,000	220,000	2,400,000
	Replicate #3	10,000	20,000	380,000	2,420,000
40	Replicate #1	10,000	10,000	30,000	180,000	94.59
	Replicate #2	10,000	10,000	40,000	240,000
	Replicate #3	10,000	10,000	20,000	200,000
75	Replicate #1	10,000	10,000	10,000	40,000	99.04
	Replicate #2	10,000	10,000	60,000	30,000
	Replicate #3	10,000	10,000	20,000	40,000
100	Replicate #1	10,000	20,000	10,000	30,000	99.30
	Replicate #2	10,000	20,000	20,000	20,000
	Replicate #3	10,000	10,000	10,000	30,000

In aqueous solutions glycolide significantly decreases pH. The following pH values were measured during the study:

pH at test start:

concentration:

10 mg/L       7.39

20 mg/L       6.74

40 mg/L       5.16

75 mg/L       3.96

100 mg/L       3.70

pH at the end of exposure

10 mg/L       7.63

20 mg/L       7.55

40 mg/L       7.37

75 mg/L       7.39

100 mg/L       7.29

The inhibitory effect is likely caused by the acidification of the growth medium. However, the growth inhibitory effect was reduced in the 10 and 20 mg/L incubation groups in which the pH returned to physiological values during the observation period.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The present study was conducted according to OECD Test Guideline 201, the green alga Selenastrum capriconutum was incubated with nominal concentrations of 0, 10, 20, 40, 75 and 100 mg/L glycolic acid (70.81%) for 72h under static conditions. Just before exposure and after 72 h exposure time the cells were counted in order to determine the growth rate. There was an inhibitory effect of glycolic acid of more than 10% in concentrations of 20 mg/L and above, thus, glycolic acid is considered to be algistatic at nominal concentrations less than or equal to 100 mg/L. The NOEC of 20 mg/L and an EC50 of 44.0 mg/L for the growth rate was determined. However, the algistatic effect is most likely caused by the strong acidification of the medium at concentrations higher than 20 mg/L. Thus, the determined EC50 and NOEC are established based on a worst case scenario.