esterification product of 2-hydroxypropane-1,3-diyl-bis(2-methylprop-2-enoate) and 3-hydroxypropane-1,2-diyl-bis(2-methylprop-2-enoate) and diphosphorus pentoxide

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:

16 July - 09 August 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study conducted under GLP with measured exposure concentrations

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, December 14, 2000

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

1224866-76-5

Cas Number:

1224866-76-5

IUPAC Name:

1224866-76-5

Details on test material:

- Name of test material (as cited in study report): Glypho
- Substance type: pure active substance
- Physical state: liquid
- Stability under test conditions: Stable
- Storage condition of test material: Dessication, darkness, 2-8°C

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Sampling method: Duplicate 4-mL samples were taken from all test concentrations and the control at time 0 and at 72-hours. At 72-hours, algae-containing replicates were pooled within each concentration before sampling.
Sample storage conditions before analysis: Samples were stored in a freezer until analysis.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: WAFs prepared at loading rates of 10, 18, 32, 56 and 100 mg/L in the test medium. Each WAF prepared separately by applying two days of magnetic stirring followed by not stirring for one hour to stabilize. The clear and colorless WAF were taken out by siphoning and used as test concentrations.

- Evidence of undissolved material (e.g. precipitate, surface film, etc): Final test solutions were clear and colorless.

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture
- Age of inoculum (at test initiation): Three days before the start of the test, cells from the algal stock culture were inoculated in culture medium. This pre-culture was maintained under the same conditions as used in the test.
- Method of cultivation: Stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21- 24°C.
ACCLIMATION
- Acclimation period: None
- Culturing media and conditions (same as test or not): Pre-culture conditions were the same
- Any deformed or abnormal cells observed: not reported

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

24 mg CaCO3/L

Test temperature:

21.1 - 23.4°C

pH:

7.1 - 8.3

Dissolved oxygen:

not applicable

Salinity:

not applicable

Nominal and measured concentrations:

Nominal WAF loading: 10, 18, 32, 56, 100 mg/L
Mean measured concentrations: < LOQ, 3.7, 7.6, 17, 30, 49 mg/L
See Table 1

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL all-glass
- Type (delete if not applicable): open / closed: capped but not sealed
- Material, size, headspace, fill volume: 50 mL of test solution
- Aeration: Continuous shaking
- Renewal rate of test solution (frequency/flow rate): None
- Initial cells density: ca. 1.0E+04 cells/mL
- Control end cells density: 135.8E+04 cells/mL
- No. of vessels per concentration (replicates): Three
- No. of vessels per control (replicates): Six plus one abiotic control at the highest concentration

GROWTH MEDIUM
- Standard medium used: Yes, test conducted in M2 medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-Q water (tap water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon and ion-exchange cartridges: Milli-Q water; Millipore Corp., Bedford, Mass., USA)
- Culture medium different from test medium: Cultures maintained on M1 medium but passaged for 72 hours in M2 before test.

OTHER TEST CONDITIONS
- Sterile test conditions: Not reported
- Adjustment of pH: None
- Photoperiod: Continuous light
- Light intensity and quality: TLD-lamps of the type ‘Cool-white’ of 30 Watt, with a light intensity within the range of 75 to 86 mE.m-2.s-1.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Counting chamber at test initiation; spectophotometer all other observations periods.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8

- Range finding study: Yes
- Test concentrations: Loading rates of 10% of 1.0, 1.0, 10, and 100 mg/L
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

yes

Remarks:

K2Cr2O7 at 0.18, 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 49 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% C.I. Not calculable

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

30 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: NOEC for biomass was also 30 mg/L

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Not calculable

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

56 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: NOEC for biomass was also 56 mg/l

Details on results:

- Exponential growth in the control (for algal test): Yes
- Observation of abnormalities (for algal test): None observed. All cells appeared normal.
- Any stimulation of growth found in any treatment: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Test substance chromatographs showed a number of peaks. Concentration was monitored using peaks 7, 9 and 25. Integrated peak areas were stable over the test period for peaks 7 and 9, but declined for peak 25. This decline was essentially identical for both the abiotic test vessel and biotic vessels (Table 1). Substance peaks were not identified and toxicity could not be assigned to any specific fraction. Therefore, it was decided to base the effect parameters on both the average concentrations calculated from the measurements on peak 25 and on the initial loading rates.

- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes. Results were found to be within the historical range for this laboratory.
- EC50 for K2Cr2O7 (reference substance): 72-hour ErC50 = 2.3 (1.9 - 2.9) mg/L (growth rate)

Reported statistics and error estimates:

Determination of calibration curve for spectrophotometric determination of cell count: Based on a calibration curve. Cell density was plotted versus extinction using spectrophotometric measurements of a minimum of six dilutions of an algal suspension with different cell densities. The calibration curve was composed using linear regression. The software automatically calculates the cell densities (Table 2) based on this curve for the spectrophotometric measurements at the various points in time during the test period.

For determination of the NOEC and the EC50 the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant reduction of growth rate (Table 3) (ANOVA, Bonferroni t-Test, TOXSTAT Release 3.5, 1996, D.D. Gulley, A.M. Boelter, H.L. Bergman). Statistically significant reduction of growth rate was found at the highest test concentration of 49 mg/l (Bonferroni t-Test, α = 0.05), however neither 10% nor 50% effect were reached at this concentration. Neither EC10 nor EC50 could be derived from the data.

Any other information on results incl. tables

Table 1, Concentrations of the test substance based on peak 25 in test medium - final test
Time of sampling [hours]^1	Loading rate^2 [mg/l]	Concentration analysed [mg/l]	Relative to initial [%]
0	0	n.d.
	0	n.d.
	10	8.93
	10	8.71
	18	14.1
	18	13.9
	32	29.7
	32	29.8
	56	52.8
	56	52.9
	100	89.3
	100	89.8
	100^4	92.3
	100^4	91.9
72	0	n.d.	n.a.
	0	n.d.	n.a.
	10	1.62^3	18
	10	1.48^3	17
	18	4.18^3	30
	18	4.20^3	30
	32	9.70	33
	32	9.78	33
	56	16.7	32
	56	16.5	31
	100	26.7	30
	100	26.7	30
	100^4	28.0	30
	100^4	28.0	30
1, Samples were stored in the freezer (≤ -15°C) until the day of analysis. 2, A water soluble fraction (WSF) prepared at the loading rate. 3, Obtained by extrapolation of the calibration curve. 4, Without algae (abiotic). n.d., Not detected. (MDL, 1.2 mg/L) n.a., Not applicable.

Table 2, Individual cell densities
Loading rate^1 Glypho (mg/l)	Vessel number	Cell density (x 1E+04) at exposure time:
		0 hours²	24 hours	48 hours	72 hours
control	1	1.00	4.62	30.85	138.37
	2	1.00	4.11	30.37	134.67
	3	1.00	3.71	29.41	119.06
	4	1.00	4.53	33.35	137.05
	5	1.00	4.42	32.16	142.62
	6	1.00	4.49	34.59	142.94
10 (3.7)	1	1.00	5.42	35.05	142.57
	2	1.00	4.97	38.26	148.15
	3	1.00	4.40	30.31	130.41
18 (7.6)	1	1.00	4.36	33.02	140.97
	2	1.00	4.44	29.11	114.36
	3	1.00	4.29	32.28	133.82
32 (17)	1	1.00	4.92	30.36	152.08
	2	1.00	4.01	29.81	141.98
	3	1.00	4.70	33.31	142.56
56 (30)	1	1.00	4.07	26.15	118.64
	2	1.00	4.61	28.12	124.65
	3	1.00	5.04	27.91	115.75
100 (49)	1	1.00	4.33	18.75	88.81
	2	1.00	4.45	25.14	104.03
	3	1.00	4.35	23.35	101.02
^1WAF prepared at the given loading rate.  Average concentration, measured, is given in parentheses. ² Number of inoculated cells at t=0: 1E+04 cells/ml

Table 3,Calculation of growth rate and rate reduction
Loading rate¹ (mg/l)	Vessel number	Growth rate (µ)				Growth rate reduction (%)
		0-24 h	24-48 h	48-72 h	0-72 h	0-24 h	24-48 h	48-72 h	0-72 h
control	1	0.06372	0.07916	0.06253	0.06847	─	─	─	─
	2	0.05886	0.08337	0.06205	0.06809	─	─	─	─
	3	0.05459	0.08629	0.05827	0.06638	─	─	─	─
	4	0.06291	0.08321	0.05889	0.06834	─	─	─	─
	5	0.06190	0.08270	0.06207	0.06889	─	─	─	─
	6	0.06253	0.08512	0.05912	0.06892	─	─	─	─
	mean	0.06075	0.08331	0.06049	0.06818	─	─	─	─
	CV	6%	3%	3%	1%	─	─	─	─
	Section CV ²	17%
10 (3.7)	1	0.07043	0.07777	0.05846	0.06889	-16	7	3	-5
	2	0.06678	0.08507	0.05641	0.06942	-10	-2	7	-9
	3	0.06176	0.08038	0.06080	0.06765	-2	4	-1	4
18 (7.6)	1	0.06135	0.08436	0.06048	0.06873	-1	-1	0	-4
	2	0.06215	0.07831	0.05702	0.06582	-2	6	6	16
	3	0.06069	0.08408	0.05925	0.06801	0	-1	2	1
32 (17)	1	0.06640	0.07581	0.06713	0.06978	-9	9	-11	-12
	2	0.05785	0.08360	0.06504	0.06883	5	0	-8	-5
	3	0.06444	0.08164	0.06058	0.06889	-6	2	0	-5
56 (30)	1	0.05849	0.07750	0.06301	0.06633	4	7	-4	13
	2	0.06370	0.07532	0.06204	0.06702	-5	10	-3	8
	3	0.06738	0.07133	0.05927	0.06599	-11	14	2	15
100 (49)	1	0.06105	0.06110	0.06479	0.06231	0	27	-7	35
	2	0.06219	0.07215	0.05918	0.06451	-2	13	2	24
	3	0.06130	0.06997	0.06103	0.06410	-1	16	-1	26
¹ WAF prepared at the given loading rate.  Average measured exposure concentration given in parantheses ² Mean CV for section-by-section specific growth rate

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

Control specific growth rate >0.92/day (0.06818/hr or 1.63/day), mean CV for section-specific growth rates in control cultures <35% (17%), CV for average specific growth rates over whole test period in replicate control cultures did not exceed 7% (1%)

Conclusions:

The 72-hour NOECgrowth rate (OECD TG 201) of the test substance to Pseudokirchneriella subcapitata is 30 mg/L based on measured substance concentration or 56 mg/L based on nominal loading rate. The EC10 and EC50 could not be determined since less than 10% inhibition was observed; there are reported as >49 mg/L based on measured substance concentration or >100 mg/L based on nominal loading rate.

Executive summary:

Toxicity of the test substance to Pseudokirchneriella subcapitata was assessed according to OECD TG203 under GLP criteria. A number of peaks were present on chromatograms of the test substance. Analytical recovery of test substance based on peak 25 declined by the end of the test, whereas other major peaks maintained stable recovery. It was decided to base the effect parameters on both the geometric mean concentrations calculated from the measurements on peak 25 and on the initial loading rates. A statistically significant effect on growth rate less than 10% was seen in the highest concentration tested. The 72-hour NOEC for growth rate was 30 mg/L based on measured substance concentration or 56 mg/L based on nominal loading rate. The EC10 and EC50 could not be determined but are >49 mg/L based on measured substance concentration or >100 mg/L based on nominal loading rate.

The test was conducted according to internationally accepted test guidelines and under GLP criteria. It is considered reliable without restriction and is suitable for Risk Assessment, Classification & Labelling, and PBT Assessment.