Reaction mass of oxydiethylene dibenzoate and oxydipropyl dibenzoate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7 November 1997 - 11 November 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OTS 797.1050 (Algal Toxicity, Tiers I and II)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Lot #: 56635211
Expiry date: 17 December 1997
Composition of sample:
Dipropylene glycol dibenzoate, CAS # 27138-31-44, 89.4%
Dipropylene glycol monobenzoate, CAS # 32686-95-6, 4.98%
Propylene glycol dibenzoate, CAS #, 19224-26-1, 2.29%
Propylene glycol monobenzoate, CAS # 37086-84-3, 0.28%
Propeneyloxy propyl benzoate, CAS # unknown, 2.35%

Analytical monitoring:

yes

Details on sampling:

- Concentrations Nominal loadings: 0 (control), 0.10, 0.22, 0.46, 1.0, 2.2, 4.6 and 10 mg/L
- Sampling method: Duplicate samples (200 mL) were taken from control and test cultures at 0 hours and at 96 hours (replicates pooled) for analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The method ofpreparation was selected following advice given in ECETOC 1996 Monograph No 26 as the test material has poor water solubility.
A stock solution of test material in chloroform (100 mg/mL) was prepared , then further stock solutions diluted using chloroform as required to give stock solutions at 46, 22, 10, 4.6, 2.2 and 1.0 mg/mL. 150 µL aliquots of each stock solution were added to autoclaved 2L vessels and the solvent was allowed to evaporate. 1.5 litres of algal medium were added to each vessel, then stirred for at least 18 hours using a magnetic stirrer with a 10 cm magnetic follower stirring at approximately 250 revolutions/minute. After stirring, the solutions were allowed to settle for approximately 1 hour, then a 500 mL (or 1 L for 1.0 mg/L level) aliquot of the water accomodated fraction was removed via a side tap from a mid water position.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): None reported

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Unicellular green algae
- Strain: No. CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae & Protozoa, Institute of Freshwater Ecology, Cumbria, UK
- Method of cultivation: Sterile nutrient medium was inoculated from a master culture and cultured under continuous illumination (c. 7000 lux) in an orbital incubator at 22°C to give an algal suspension in log phase growth, characterised by a cell density of 1.4E+07 cells / mL


ACCLIMATION
- Acclimation period:
- Culturing media and conditions (same as test or not): Lighting conditions used were equivalent to main test; temperature specification was slightly
different (22°C, c.f. 24±1°C in main test).
- Any deformed or abnormal cells observed: None reported

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

24 ± 1°C

pH:

7.3 to 7.4

Dissolved oxygen:

Not quantified - gaseous exchange was ensured by the action of the orbital shaker, oscillating at 120 cycles per minute.

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal loadings: 0 (control), 0.10, 0.22, 0.46. 1.0, 2.2, 4.6, 10 mg/L
Geometric mean measured concentrations for dipropylene glycol dibenzoate: 0.070, 0.16, 0.27, 0.68, 1.6, 3.2 and 5.9 mg/L See Table 1

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed (loosely stoppered flasks)
- Material, size, headspace, fill volume: 250 mL flasks containing 100 mL medium.
- Aeration: Gaseous exchange and suspension of the algal cells were ensured by the action of the orbital shaker, oscillating at 120 cycles per minute.
- Renewal rate of test solution (frequency/flow rate): None (static test)
- Initial cells density: 1 x 10^4 cells/mL nominal, control cell density at 0h was 1.48 x 10^4 cells/mL.
- Control end cells density: 1.77 x 10^6 cells/mL
- No. of vessels per concentration (replicates):3
- No. of vessels per control (replicates):3


GROWTH MEDIUM
- Standard medium used: yes (Sterile nutrient medium as recommended in Official Journal No. L383A Part C3).


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Prepared in-house using deionised / reverse osmosis water.


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: Not specified
- Photoperiod: Continuous illumination
- Light intensity and quality: 7000 lux


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter. Measurements taken at 0, 24, 48, 72, and 96 hours.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: Approximately 2.2
- Range finding study
- Test concentrations: range finding studies conducted with initial loading rates of test substance ranging from 0.01 to 10 mg/L.
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

95% CI 2.9, 3.7 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.68 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.64 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

95% CI 0.46,0.81 mg/L

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

1.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

95% CI 0.89,1.4 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other:

Remarks:

95% CI 0.57, 0.86 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.16 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

2.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

95% CI 2.3, 2.6 mg/L

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.27 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

96 h

Dose descriptor:

EC10

Effect conc.:

0.66 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

95% CI 0.57, 0.74 mg/L

Duration:

96 h

Dose descriptor:

EC20

Effect conc.:

1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

95% CI 0.94,1.1 mg/L

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

0.62 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other:

Remarks:

95% CI 0.53, 0.73 mg/L

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

0.07 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

96 h

Dose descriptor:

EC10

Effect conc.:

0.12 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other:

Remarks:

95% CI 0.079, 0.15 mg/L

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

0.15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

0.89 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

96 h

Dose descriptor:

EL10

Effect conc.:

0.19 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

96 h

Dose descriptor:

EL10

Effect conc.:

0.87 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

1.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

4.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

96 h

Dose descriptor:

EL50

Effect conc.:

0.95 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

96 h

Dose descriptor:

EL50

Effect conc.:

3.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

0.22 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

96 h

Dose descriptor:

NOELR

Effect conc.:

0.46 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No abnormalities were detected in any of the cultures examined. Bacterial contamination, observed in some of the test cultures at low levels, was not considered significant. The presence of bacteria during the study is thought to be due to the test substance being a biodegradable substrate for bacteria.
- Any stimulation of growth found in any treatment: A slight increase in growth rate was seen in the 0.10, 0.22 and 0.46 mg/L cultures at 72 hours, and in the 0.22 and 0.46 mg/L cultures at 96 hours.
The analytical method used to confirm the concentration of test material in solution determined concentrations of dipropylene glycol monobenzoate DPGMB) as well as dipropylene glycol dibenzoate (DPGDB); the concentration of DPGDB was found to be lower after 96 hours than at the start of the test in all cases, whereas the concentration of DPGMB had increased in all samples. This observation suggests that the monobenzoate was a degradation product of the dibenzoate.

Reported statistics and error estimates:

The original study report determined the endpoints based upon nominal loading rates only. Subsequent to the study the endpoints were re-calculated by the performing laboratory based upon the geometric mean measured concentrations. The ECx values and associated confidence limits for growth rate and biomass integral were calculated by Probit analysis using Linear Maximum-Likelihood regression. All statistical analyses were performed using the ToxRatPro software package (TOXRAT, 2001-2018). The No Observed Effect Concentration (NOEC) values were determined using the Williams' statistical test based on the original statistical analysis of the nominal loading rates (NOELRs) associated with their coresponding calculated mean measured concentrations.

Table 1: Initial (0 hours), Final (96 hours), and Geometric Mean Measured Concentrations

Dipropylene Glycol Dibenzoate				Dipropylene Glycol Monobenzoate
Nominal Loading Rate (mg/L)	Initial Conc (mg/L)	Final Conc (mg/L)	Geometric Mean Conc. (mg/L)	Initial Conc (mg/L)	Final Conc (mg/L)	Geometric Mean Conc. (mg/L)
Control	0.001289	ND	-	ND	ND	ND
0.1	0.09909	0.0501	0.07	0.006097	0.0235	0.01
0.22	0.2125	0.1152	0.16	0.01388	0.05007	0.03
0.46	0.3764	0.1874	0.27	0.02216	0.09337	0.05
1	0.8755	0.5331	0.68	0.05528	0.195	0.10
2.2	1.929	1.321	1.60	0.1234	0.316	0.20
4.6	3.363	3.038	3.20	0.2331	0.4955	0.34
10	6.858	5.125	5.93	0.05732	0.7783	0.21

Table 2: Cell densities and phys-chem measurements

Nominal initial loading rates (mg/L)		pH	Cell densities (cells/ml)					pH
		0 hour	0 hour	24 hours	48 hours	72 hours	96 hours	96 hours
Control	R1	7.6	1.36E+04	1.65E+04	7.93E+04	3.82E+05	1.70E+06	7.3
	R2	7.6	1.80E+04	1.58E+04	7.30E+04	4.30E+05	1.85E+06	7.3
	R3	7.6	1.27E+04	1.59E+04	8.28E+04	4.15E+05	1.75E+06	7.3
	Mean	7.6	1.48E+04	1.61E+04	7.83E+04	4.09E+05	1.77E+06	7.3
0.1	R1	7.7	1.16E+04	1.64E+04	2.53E+04	3.46E+05	1.38E+06	7.3
	R2	7.7	1.20E+04	1.40E+04	1.94E+05	3.42E+05	1.43E+06	7.3
	R2	7.7	1.16E+04	1.41E+04	6.71E+04	3.64E+05	1.46E+06	7.3
	Mean	7.7	1.17E+04	1.48E+04	9.53E+04	3.51E+05	1.42E+06	7.3
0.22	R1	7.7	1.22E+04	1.54E+04	3.16E+04	3.51E+05	1.38E+06	7.4
	R2	7.7	1.10E+04	1.44E+04	2.56E+04	3.61E+05	1.36E+06	7.4
	R3	7.7	1.07E+04	1.71E+04	6.45E+04	3.81E+05	1.51E+06	7.4
	Mean	7.7	1.13E+04	1.56E+04	4.05E+04	3.64E+05	1.42E+06	7.4
0.46	R1	7.7	1.09E+04	1.45E+04	3.11E+04	3.36E+05	1.35E+06	7.3
	R2	7.7	1.01E+04	1.45E+04	2.48E+04	3.51E+05	1.33E+06	7.3
	R3	7.7	1.03E+04	1.39E+04	2.59E+04	3.37E+05	1.37E+06	7.3
	Mean	7.7	1.04E+04	1.43E+04	2.73E+04	3.41E+05	1.35E+06	7.3
1.0	R1	7.7	1.02E+04	1.38E+04	2.78E+04	2.62E+05	8.89E+05	7.3
	R2	7.7	1.19E+04	1.41E+04	2.83E+04	2.60E+05	9.13E+05	7.3
	R3	7.7	1.16E+04	1.40E+04	4.07E+04	2.82E+05	1.02E+06	7.3
	Mean	7.7	1.12E+04	1.40E+04	3.23E+04	2.68E+05	9.41E+05	7.3
2.2	R1	7.7	1.05E+04	1.38E+04	3.90E+04	1.03E+05	2.39E+05	7.3
	R2	7.7	9.85E+03	1.35E+04	3.54E+04	1.01E+05	2.61E+05	7.3
	R3	7.7	1.52E+04	1.44E+04	2.75E+04	1.05E+05	2.67E+05	7.3
	Mean	7.7	1.19E+04	1.39E+04	3.40E+04	1.03E+05	2.56E+05	7.3
4.6	R1	7.8	1.00E+04	1.31E+04	3.02E+04	4.37E+04	5.79E+04	7.4
	R2	7.8	9.90E+03	1.25 E+04	3.70E+04	5.21E+04	6.16E+04	7.4
	R3	7.8	1.00E+04	1.31E+04	3.36E+04	5.33E+04	7.04E+04	7.4
	Mean	7.8	1.00E+04	1.29E+04	3.36E+04	4.97E+04	6.33E+04	7.4
10	RI	7.8	1.12E+04	1.22E+04	2.37E+04	4.19E+04	2.96E+04	7.4
	R2	7.8	9.69E+03	1.24E+04	1.82E+04	3.62E+04	3.06E+04	7.4
	R3	7.8	1.22E+04	1.24E+04	1.82E+04	3.06E+04	4.00E+04	7.4
	Mean	7.8	1.10E+04	1.23E+04	2.00E+04	3.62E+04	3.34E+04	7.4
Temperature °C		--	24	24	24	24	24	--

Table 3: Results based upon geometric mean measured concentrations

Time Point (hours)	Parameter Evaluated	Endpoint	mg/L (95% CI)
72	Growth Rate	ErC10 (95% Confidence Interval)	0.64 (0.46 to 0.81)
		ErC20 (95% Confidence Interval)	1.1 (0.89 to 1.4)
		ErC50 (95% Confidence Interval)	3.2 (2.9 to 3.7)
		No Observable Effect Concentration (NOEC)	0.68
72	Biomass	ErC10 (95% Confidence Interval)	0.081 (0.049 to 0.12)
		ErC20 (95% Confidence Interval)	0.17 (0.12 to 0.23)
		ErC50 (95% Confidence Interval)	0.70 (0.57 to 0.86)
		No Observable Effect Concentration (NOEC)	0.16
96	Growth Rate	ErC10 (95% Confidence Interval)	0.66 (0.57 to 0.74)
		ErC20 (95% Confidence Interval)	1.0 (0.94 to 1.1)
		ErC50 (95% Confidence Interval)	2.5 (2.3 to 2.6)
		No Observable Effect Concentration (NOEC)	0.27
96	Biomass	ErC10 (95% Confidence Interval)	0.12 (0.079 to 0.15)
		ErC20 (95% Confidence Interval)	0.21 (0.16 to 0.26)
		ErC50 (95% Confidence Interval)	0.62 (0.53 to 0.73)
		No Observable Effect Concentration (NOEC)	<0.070

Validity criteria fulfilled:

yes

Conclusions:

The original study report provide the endpoints based upon the nominal loading rates. The EL50 (Area under the curve 72 h) was 1.1 mg/L and the EL50 (Growth rate 0-72 h was 4.9 mg/L while the EL50 (Area under the curve 96 h) was 0.95 mg/L and the EL50 (Growth rate 0-96 h) was 3.6 mg/L. The exposure solutions were analytically measured and subsequent to the study the performing labortory recalculated the endpoints based upon the geometric mean measured concentrations. The 72 and 96 hour ErC50s (growth rate) were determined to be 3.2 and 2.5 mg/L respectively. The 72 and 96 hour NOECs for growth rate were determined to be 0.68 and 0.27 mg/L respectively.

Executive summary:

An algal growth inhibition test was conducted to determine the effect on the growth of algae of the test material dipropylene glycol dibenzoate (DPGDB). The study was conducted according to EC, OECD, and US EPA test guidelines, and in compliance with GLP.

The exposure solutions were analytically measured and subsequent to the study the performing labortory recalculated the endpoints based upon the geometric mean measured concentrations.  The 72 and 96 hour ErC50s (growth rate) were determined to be 3.2 and 2.5 mg/L respectively.  The 72 and 96 hour NOECs for growth rate were determined to be 0.68 and 0.27 mg/L respectively.

Reference 2

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material:
Manufactured by: Eastman Chemical Company
Manufacture Date: 01/08/2020
Batch #: V245001001
Expiry Date: 01/08/2022

- Purity, including information on contaminants, isomers, etc.: 93.113%
GC analysis by area percent:
93.113 Diethylene glycol dibenzoate
3.982 Diethylene glycol monobenzoate
0.714 Dipropylene glycol dibenzoate isomers (3)
0.709 Ethylene glycol dibenzoate
0.518 2-Hydroxyethyl benzoate
0.294 2-(2-Benzyloxy)ethoxy)propyl benzoate
0.121 Di-2-ethylhexyl terephthalate
0.085 Triethylene glycol dibenzoate
99.536 Total


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark

Analytical monitoring:

yes

Details on sampling:

- Concentrations: A nominal amount of test item (150 mg) was dispersed in 3 L of culture medium with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 L used to pre condition the filter was discarded) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give stock solutions of 18, 10, 5.6, 3.2, 1.8 and 1.0% v/v saturated solution.
- Sampling method: The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0, 24, 48 and 72 hours. Samples were taken from the inoculated control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for immediate quantitative analysis. Further samples of each inoculated test group were incubated alongside the test to provide samples for analysis at 24 and 48 hours.
In order to determine whether losses occurred due to adsorption of the test item to the algal cells present, samples of each test group, to which no algal cells were added, were incubated alongside the test to provide samples for analysis at 24, 48 and 72 hours. Duplicate samples were taken at each occasion and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Based upon previous studies the test material was considered to be a "difficult substance" as defined by the OECD Guidance Document on Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals (OECD 2019). Therefore a media preparation trial was conducted in order to determine the solubility of the test item under test conditions.

An initial Media Preparation Trial was conducted by dispersing a 300 mg nominal amount of test item, in duplicate, in 3L of algal culture medium and stirred at approximately 1500 rpm for periods of either 24 or 48 hours. After stirring the samples were taken for chemical analysis after the following pre-treatments:
- Filtration through 0.2 um Gelman Acrocap filter (approximately 100 ml used to pre-condition the filter was discarded)
- Filtration through 0.2 um Gelman Acrocap filter (approximately 500 ml used to pre-condition the filter was discarded)
Observations made on the preparations after filtration showed they were very slightly hazy dispersions indicating that some undissolved test item has passed through the filter. Given this a further media preparation trial was conducted using a lower initial loading rate and an alternative filter type in an attempt to prevent undissolved test item passing through the filter.

Further Media Preparation Trial
A nominal amount of test item (250 mg) was dispersed, in duplicate, in 5 L of culture medium with the aid of propeller stirring at approximately 1500 rpm for periods of either 24 or 48 hours. After stirring samples were taken for chemical analysis after the following pre treatments:
• Filtration through a 0.2 μm Sartorious Sartopore filter (approximately 1 L used to pre condition the filter was discarded)
• Filtration through a 0.2 μm Sartorious Sartopre filter (approximately 2 L used to pre condition the filter was discarded)
Observations made on the preparations after filtration showed they were all clear colorless solutions. No increase in the dissolved test item concentration obtained occurred when the preparation period was extended beyond 24 hours. Based on this information the test item was prepared using a saturated solution method of preparation at an initial loading rate of 50 mg/L, stirred for a period of 24 hours prior to the removal of any undissolved test item by filtration through a 0.2 μm Sartorious Sartopore filter (first approximate 2 L discarded) to give a 100% v/v saturated solution of the test item.

Solution Preparation in Definitive Test
A nominal amount of test item (150 mg) was dispersed in 3 L of culture medium with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 L used to pre condition the filter was discarded) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give stock solutions of 18, 10, 5.6, 3.2, 1.8 and 1.0% v/v saturated solution. An aliquot (1000 mL) of each of the stock solutions was separately inoculated with 4.5 mL of algal suspension to give an initial nominal cell density of 5.00 x 103 cells/mL. The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland
- Age of inoculum (at test initiation): Approximately 3 to 4 days before the start of the test, inoculum cultures of algae were set up at an initial cell density of approximately 10^3 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ± 1C until the algal cell density was approximately 10^5 to 10^6 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

Temperature was maintained at 24 ± 1 ºC throughout the test.

pH:

The pH value of the control cultures was observed to increase from pH 7.4 at 0 hours to pH 9.0 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 CO2 from the gaseous phase to the aqueous phase. In this situation CO2 required 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 Test Guideline (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.

Nominal and measured concentrations:

Analysis of the test preparations to which algal cells had been added showed measured test concentrations at 0 hours to range from 0.29 to 5.6 mg/L. A concentration dependent decline in measured test concentration was observed after each 24-Hour period in the range of 0.28 to 5.1 mg/L at 24 hours, 0.060 to 4.5 mg/L at 48 hours and from less than the limit of quantification (LOQ) of the analytical method employed, determined to be 0.011 mg/l, to 1.9 mg/L at 72 hours.
In order to determine whether the decline in measured test concentrations observed was attributable to adsorption of the test item to the algal cells present, additional samples to which no algal cells were added were prepared at test initiation and incubated alongside the test. Samples provided for analysis at 24, 48 and 72 hours showed measured test concentrations to range from 0.29 to 5.4 mg/L at 24 hours, from 0.26 to 5.4 mg/L at 48 hours and from 0.26 to 5.2 mg/L at 72 hours. These results demonstrate that the test item was stable under the conditions of the test and therefore the losses observed in those samples to which algae were added was attributable to adsorption only.
Given that the test item was shown to be stable under test conditions, it was considered appropriate to calculate the results based on the measured test concentrations obtained from the preparations which contained no algal cells as whilst losses through adsorption were demonstrated it can still be considered that the algae were exposed to the test item. As the measured concentrations obtained from the uninoculated preparations at 24, 48 and 72 hours were all within 80% to 120% of the 0-Hour measured concentrations it was considered justifiable to calculate the results based on the 0-Hour measured test concentrations only (see Table 1 below).

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass conical flasks
- Material, size, headspace, fill volume: 100 mL plugged with polyurethane foam bungs
- Renewal rate of test solution (frequency/flow rate): static (no renewal)
- Initial cells density: 5.00 x 10^3 cells per mL
- Control end cells density: 1.20 x 10^6 cells per mL
- 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 culture medium was prepared using reverse osmosis purified deionized water and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.
- Intervals of water quality measurement: 24 hours

OTHER TEST CONDITIONS
- Photoperiod: Continuous
- Light intensity and quality: Intensity approximately 7000 lux provided by warm white lighting (380 to 730 nm)
- Mixing: constantly shaken at approximately 150 rpm for 72 hours


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] Samples were taken at 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample. The nominal inoculated cell concentration (5.00 x 10^3 cells/mL) was taken as the starting cell density. 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.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Approximately 1.8
- Range finding study: yes
- Test concentrations (range-finding): 0.10, 1.0, 10, 100% v/v saturated solution
- Results used to determine the conditions for the definitive study: The results showed no effect on growth at the test concentrations of 0.10 and 1.0% v/v saturated solution. However, growth was observed to be reduced at 10 and 100% v/v saturated solution.
Test Concentrations (definitive study): 1.0, 1.8, 3.2, 5.6, 10, 18% v/v saturated solution

Reference substance (positive control):

yes

Remarks:

A positive control test using potassium dichromate as the reference item is performed twice in a 12 month period to demonstrate satisfactory conditions of the test.

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.6 mg/L

95% CI:

1.4 - 1.8

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Final measurements of concentration were within 20% of initial and therefore results based upon initial measurements

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

2.1 mg/L

95% CI:

1.9 - 2.3

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3.7 mg/L

95% CI:

3.5 - 3.9

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.54 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.98 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.48 mg/L

95% CI:

0.23 - 0.7

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.7 mg/L

95% CI:

0.41 - 0.93

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.4 mg/L

95% CI:

1.1 - 1.8

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.29 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): none observed
- Adherence to test vessels: not observed
- Aggregation of algal cells: not observed
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Differences in measurements of solutions with and without algal cells indicated absorption of test material to algal cells. Measurements made in replicate flasks without algal cells represent exposure concentrations and were used for endpoint calculations
- Effect concentrations exceeding solubility of substance in test medium: no

Validity criteria fulfilled:

yes

Executive summary:

1.1                   Introduction

A study was performed to assess the effect of the test item on the growth of the green alga Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata).  The method followed that described in 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) No 761/2009.

1.2                   Methods

Information provided by the Sponsor indicated the water solubility of the test item to be approximately 38 mg/L.  Preliminary media preparation trials indicated that the use of a saturated solution method of preparation was most appropriate for this test item.

Following a preliminary range‑finding test and initial experiments, Raphidocelis subcapitata was exposed to aqueous solutions of the test item at nominal concentrations of 1.0, 1.8, 3.2, 5.6, 10 and 18% v/v saturated solution (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C.  The test item solutions were prepared by stirring an excess (50 mg/L) of test item in culture medium using a propeller stirrer at approximately 1500 rpm for 24 hours.  After the stirring period any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 L used to pre‑condition the filter was discarded) to produce a 100% v/v saturated solution of the test item.  This saturated solution was then further diluted to give the required test concentrations of 1.0, 1.8, 3.2, 5.6, 10 and 18% v/v saturated solution.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter^® Multisizer Particle Counter.

1.3                   Results

Analysis of the test preparations to which algal cells had been added showed measured test concentrations at 0 hours to range from 0.29 to 5.6 mg/L.  A concentration dependent decline in measured test concentration was observed after each 24-Hour period in the range of 0.28 to 5.1 mg/L at 24 hours, 0.060 to 4.5 mg/L at 48 hours and from less than the limit of quantification (LOQ) of the analytical method employed, determined to be 0.011 mg/l, to 1.9 mg/L at 72 hours.

In order to determine whether the decline in measured test concentrations observed was attributable to adsorption of the test item to the algal cells present, additional samples to which no algal cells were added were prepared at test initiation and incubated alongside the test.  Samples provided for analysis at 24, 48 and 72 hours showed measured test concentrations to range from 0.29 to 5.4 mg/L at 24 hours, from 0.26 to 5.4 mg/L at 48 hours and from 0.26 to 5.2 mg/L at 72 hours.  These results demonstrate that the test item was stable under the conditions of the test and therefore the losses observed in those samples to which algae were added was attributable to adsorption only.

Given that the test item was shown to be stable under test conditions, it was considered appropriate to calculate the results based on the measured test concentrations obtained from the preparations which contained no algal cells as whilst losses through adsorption were demonstrated it can still be considered that the algae were exposed to the test item.  As the measured concentrations obtained from the uninoculated preparations at 24, 48 and 72 hours were all within 80% to 120% of the 0-Hour measured concentrations it was considered justifiable to calculate the results based on the 0-Hour measured test concentrations only.

Exposure of Raphidocelis subcapitata to the test item gave the following results based on the 0-Hour measured test concentrations:

Response Variable	Endpoint	Measured Concentration (mg/L) [95% Confidence Limits]
Growth Rate	ErC10	1.6 [1.4 to 1.8]
	ErC20	2.1 [1.9 to 2.3]
	ErC50	3.7 [3.5 to 3.9]
	No Observed Effect Concentration (NOEC)	0.54
	Lowest Observed Effect Concentration (LOEC)	0.98
Yield	EyC10	0.48 [0.23 to 0.70]
	EyC20	0.70 [0.41 to 0.93]
	EyC50	1.4 [1.1 to 1.8]
	No Observed Effect Concentration (NOEC)	<0.29
	Lowest Observed Effect Concentration (LOEC)	≤0.29

 

Description of key information

Toxicity data for the reaction mass is provided from available studies of the two individual components of the reaction mass.  The key values identified for the chemical safety assessment are the most sensitive endpoints from the individual tests of the components, therefore providing a conservative assessment of the hazard for the reaction mass.  

 

DPGDB:

Toxicity to aquatic algae and cyanobacteria:

- Key study, reliability 1, OECD 201, Pseudokirchneriella subcapitata - The EbC50 (biomass, 72 h) was 0.7 mg/L and the ErC50 (Growth rat,e 0 - 72 h) was 3.2 mg/L. The EbC50 (biomass, 96 h) was 0.62 mg/L and the ErC50 (Growth rate, 0 - 96 h) was 2.5 mg/L. The corresponding 72 hour and 96 hour "no-observed effect concentration" (NOEC) for biomass were 0.16 mg/L and <0.07 mg/L, respectively. The corresponding 72 hour and 96 hour "no-observed effect concentration" (NOEC) for Growth Rate were 0.68 mg/L and 0.27 mg/L, respectively.

 

DEGDB:

Toxicity to aquatic algae and cyanobacteria:

-Key study, reliability 1, OECD 201, Raphidocelis subcapitata.  The ErC10, ErC20, and ErC50 (growth rate 0-72 hours) were 1.6, 2.1, and 3.7 mg/L respectively.  The EyC10, EyC20, and EyC50 (yield 72 hours) were 0.48, 0.70, and 1.4 mg/L respectively.

- Disregarded study, reliability 3, OECD 201, Pseudokirchneriella subcapitata - The 72h EL50 (Area under the curve, biomass) was 5.2 mg/L and the 72h EL50 (Growth rate) was 11 mg/L. The corresponding "No-observed effect loading rate" (NOELR) were 2.2 mg/L and 1.0 mg/L, respectively. The 96h EL50 (Area under the curve, biomass) was 5.9 mg/L and the 96h EL50 (Growth rate) was 15 mg/L. The corresponding NOELR for both were 2.2 mg/L. Study endpoints were based upon nominal loading rates. Analytical measurement of the exposure solutions were made, however the final measurements were all at non-detect levels resulting in the geometric mean measured concentrations being unreliable. The study was determined to be unreliable and was repeated.

Key value for chemical safety assessment

EC50 for freshwater algae:

3.2 mg/L

EC50 for marine water algae:

3.2 mg/L

EC10 or NOEC for freshwater algae:

0.68 mg/L

EC10 or NOEC for marine water algae:

0.68 mg/L

Additional information

This substance is a reaction mass of dipropylene glycol dibenzoate (DPGDB) and diethylene glycol dibenzoate (DEGDB). No testing has been performed on the reaction mass itself, but data are available for DPGDB and DEGDB individually. All the studies were performed according to international test guidelines and in compliance with GLP.  The algal growth inhibition studies were performed on Raphidocelis subcapitata (formally known as Selenastrum capricornutum or Pseudokirchnerella subcapitata). In the initial studies the substance was introduced using a water accommodated fraction (WAF) and the endpoints were reported using nominal loading rates. The exposure solutions in these studies were analytically measured at the beginning and end of the exposures. The studies were re-evaluated based upon the measured concentrations documented in the study reports. The study of DEGDB had final analytical measurements of the exposure solutions that were at non-detect levels and therefore the geometric means of the measured concentrations are belived to be unreliable. An algal test was reconducted for this component of the reaction mass. The algal test of DPGDB provided analytical measurements of the exposure solutions that are believed to be reliable and the endpoints were recalculated based upon the geometric mean measured concentrations. All existing data suggest that the components of the reaction mass have quantitatively similar properties and the hazard assessment (PNEC derivation) for the reaction mass will be based upon a worst-case evaluation of data from the individual components. 

 

The key values are:

Short-term toxicity to aquatic algae and cynaobacteria: EC50 = 3.2 mg/L (DPGDB 72 hour ErC50)

Long-term toxicity to aquatic algae and cynaobacteria: NOEC = 0.68 mg/L(DPGDB 72 hour NOEC growth rate)