Reaction mass of oxydiethylene dibenzoate and oxydipropyl dibenzoate

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 October 1997 - 13 October 1997.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study undertaken in GLP accredited laboratory.

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OTS 797.1300 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)

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), 1.0, 2.2, 4.6, 10, 22, 46 and 100 mg/L
- Sampling method: Duplicate pooled medium samples were taken at 0 and 48 hours; sampling methodology not specified.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The method of preparation was selected following advice given in ECETOC 1996 Monograph No 26 as the test material has poor water solubility.
Test material was added by accurate pipette to 2 L glass vessels of Elendt M4 medium and stirred for at least 18 hours. Once stirred the solutions were left to stand for one hour prior to removing the water accommodated fraction. Approximately 800 mL of the solution was removed from a mid-water position.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): In preparation of the water accommodated fraction, when it was left to stand (without stirring), globules of test material were seen at the bottom of the vessels and a film was seen on the surface of the water. In the exposure vessels, expired solutions were noted to be cloudy after 24 hours.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Strain: Straus
- Source: Cultured in-house from a strain originating from the Institute National de Recherche Chimique Applique (IRChA), France
- Age at study initiation (mean and range, SD): Less than 24 hours old
- Method of breeding: The Daphnia used were bred under conditions which ensures that cultures reproduce by parthenogenesis

ACCLIMATION
- Acclimation period: Not applicable - the Daphnia used were all less than 24 hours old. Stock culture conditions are provided below.
- Type and amount of food: A suspension of the unicellular green alga, Chlorella vulgaris

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test temperature:

19 to 21°C

pH:

7.9 to 8.1

Dissolved oxygen:

7.0 to 8.0 mgO2 / L

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal loadings(fresh) ; 0 (control), 1.0, 2.2, 4.6, 10.0, 22, 46 and 100 mg/L
See Table 3 for measured concentrations of dipropylene glycol dibenzoate and monobenzoate

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type : closed (loosely covered to minimise evaporative losses)
- Material, size, headspace, fill volume: 250 mL glass jars, filled with 200 mL test medium
- Renewal rate of test solution (frequency/flow rate): None (static test)
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: 40 mL test solution per organism.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4 medium, prepared in house using analytical reagents and deionised water

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours light / 8 hours dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Immobilisation

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

5 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

43.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

19.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence limits : 13.1 - 28.5 mg/L

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

2.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 5% immobilisation was caused at 2.2 mg/L; this level of immobilisation is not considered significant.

Details on results:

- Mortality of control: Two Daphnia in the control level were immobilised after 48 hours. As this corresponded to the no-effect level of 10%, the study results were still considered valid.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Precipitation was observed, as noted previously. 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 48 hours than at the start of the test in all cases, whereas the concentration of DPGMB had generally increased. This observation suggests that the monobenzoate was a degradation product of the dibenzoate.

Reported statistics and error estimates:

48 hour EC50 = 5.0 mg/L. 95% CI 3.96 - 6.05 mg/L based upon mean measured dipropylene glycol dibenzoate concentrations. The original study report provided EC50 and NOELR results based upon nominal loading rates only. The key result EC50 was calculated post study based upon the mean measured dipropylene dibenzoate concentrations reported in the study. The EC50 was calculated using Probit Model within the TOSTAT version 3.4 statistical program.

Table 1:Cumulative Daphnia magna Immobilization Data
Loading Rate (mg/L)	Measured Conc. (mg/L)	Cumulative Immobilized (n=5)
		24 Hours						48 Hours
		R1	R2	R3	R4	Total	Mean (%)	R1	R2	R3	R4	Total	Mean (%)
Control	0	0	0	0	1	1	5	1	0	0	1	2	10
1	0.260	0	1	0	0	1	5	0	1	1	0	2	10
2.2	0.748	0	0	0	0	0	0	0	0	0	1	1	5
4.6	1.325	0	1	0	0	1	5	0	1	3	1	5	25
10	3.802	1	0	0	0	1	5	2	3	1	2	8	40
46*	5.272	4	3	2	2	11	55	4	3	3	2	12	60
22*	7.465	1	3	4	3	11	55	4	5	4	4	17	85
100	13.174	4	3	4	3	14	70	5	4	5	4	18	90
*Revised order for statistical analyses as measured concentration for 22 mg/L LR was higher than measured value for 46 mg/L LR

Table 2
Benzoflex 9-88: Environmental parameters Mean values for pH, temperature and dissolved oxygen
Nominal initial Loading rate mg/L	Mean values
	pH	mgO2/L	T°C
Control	8.0	8.0	19.3
1.0	8.1	7.5	19.7
2.2	8.1	7.5	19.7
4.6	8.0	7.4	19.7
10	7.9	7.3	19.7
22	7.9	7.1	19.7
46	7.9	7.0	19.7
100	7.9	7.1	19.7

Table 3: Loading Rate and Measured Dibenzoate and Monobenzoate Concentrations
Dibenzoate Concentrations
Loading Rate (mg/L)	Initial Conc (mg/L)	Final Conc (mg/L)	Mean Conc (mg/L)
Control	ND	ND	ND
1	0.3388	0.1814	0.2601
2.2	0.8953	0.6012	0.7483
4.6	1.415	1.235	1.3250
10	4.94	2.663	3.8015
22	8.869	6.06	7.4645
46	6.33	4.213	5.2715
100	19.27	7.077	13.1735
Monobenzoate Concentrations
Loading Rate (mg/L)	Initial Conc (mg/L)	Final Conc (mg/L)	Mean Conc (mg/L)
Control	ND	ND	ND
1	0.036	0.08989	0.062945
2.2	0.1092	0.3013	0.20525
4.6	0.2398	0.7354	0.4876
10	0.4793	1.818	1.14865
22	1.274	2.485	1.8795
46	3.501	3.804	3.6525
100	13.87	6.866	10.368

Validity criteria fulfilled:

yes

Conclusions:

The 48 hour EC50 (immobilisation value for DPGDB with Daphnia magna) was determined to be 5.0 mg/L (95% CI 3.96 - 6.05 mg/L) based upon mean measured dipropylene glycol dibenzoate concentrations.

Executive summary:

An acute Daphnia Immobilization study was performed to determine the actue toxicity 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. Due to low water solubility of the test substance, the exposure solutions were prepared as Water Accommodated Fractions (WAF) and the test results were originally reported as WAF loading rates. However, the exposure solutions were analytically measured during the study for the parent dibenzoate and breakdown monobenzoate concentrations. Subsequent to the study the endpoints were re-calculated based upon the mean measured concentrations of the parent substance, dipropylene glycol dibenzoate.

The 48 hour EC50 with Daphnia magna was determined to be 5.0 mg/L (95% CI 3.96 - 6.05 mg/L) based upon mean measured dipropylene glycol dibenzoate concentrations.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 November 1997 - 14 November 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted in accordance with EC, OECD, and US EPA test guidelines, and in complaince with GLP.

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OTS 797.1300 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Lot #: 56625030
Expiry date: 19 December 1997
Composition of sample:
Diethylene glycol dibenzoate, CAS # 120-55-8, 89.9%
Diethlene glycol monobenzoate, CAS # 20587-61-5, 5.85%
Dipropylene glycol dibenzoate, CAS #, 27138-31-44, 1.5%
Ethylene glycol dibenzoate, CAS # 94-49-5, 0.19%
Triethylene glycol dibenzoate, CAS # 120-56-9, 0.15%
Unidentified component, CAS # unknown, 0.54%
Unidentified component, CAS # unknown, 0.10%
Unidentified component, CAS # unknown, 0.10%

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Nominal loadings - 0 (control), 1.0, 2.2, 4.6, 10, 22, 46, and 100 mg/L.
- Sampling method: Duplicate pooled medium samples were taken at 0 and 48 hours; sampling methodology not specified.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Method followed advice given in ECETOC 1996, Monograph No. 26 as the test material has poor water solubility.
Test material was added by accurate pipette to 2 L glass vessels of Elendt M4 medium and stirred for at least 18 hours. Once stirred the solutions were left to stand for one hour prior to removing the water accommodated fraction. Approximately 800 mL of the solution was removed from a mid-water position.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): In preparation of the water accommodated fraction, when it was left to stand (without stirring), globules of test material were seen at the bottom of the vessels and a film was seen on the surface of the water. In the exposure vessels, expired solutions were noted to be cloudy after 24 hours.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Strain: Straus
- Source: Cultured in-house from a strain originating from the Institute National de Recherche Chimique Applique (IRChA), France
- Age at study initiation (mean and range, SD): Less than 24 hours old
- Method of breeding: The Daphnia used were bred under conditions which ensures that cultures reproduce by parthenogenesis

ACCLIMATION
- Acclimation period: Not applicable - the Daphnia used were all less than 24 hours old. Stock culture conditions are provided below.
- Type and amount of food: A suspension of the unicellular green alga, Chlorella vulgaris

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test temperature:

19.3°C

pH:

7.7 - 7.8

Dissolved oxygen:

8.1 - 9.0 mgO2/L

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal loadings: 0 (control), 1.0, 2.2, 4.6, 10, 22, 46, and 100 mg/L.
See Table 3 for measured concentrations of diethylene glycol dibenzoate

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type : closed (loosely covered to minimise evaporative losses)
- Material, size, headspace, fill volume: 250 mL glass jars, filled with 200 mL test medium
- Renewal rate of test solution (frequency/flow rate): None (static test)
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: 40 mL test solution per organism.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4 medium, prepared in house using analytical reagents and deionised water

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours light, 8 hours dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : immobilisation

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

3.049 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

6.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence limits : 4.4 - 10.0 mg/L

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 10% immobilisation was caused at 1.0 mg/L; this level of immobilisation is not considered significant.

Details on results:

- Mortality of control: Two Daphnia in the control level were immobilised after 48 hours. As this corresponded to the no-effect level of 10%, the study results were still considered valid.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Precipitation was observed, as noted previously. The analytical method used to confirm the concentration of test material in solution determined concentrations of diethylene glycol monobenzoate DEGMB as well as diethylene glycol dibenzoate (DEGDB); the concentration of DEGDB was found to be lower after 48 hours than at the start of the test in most cases, whereas the concentration of DEGMB had generally increased. This observation suggests that the monobenzoate was a degradation product of the dibenzoate.

Reported statistics and error estimates:

The 48 hour EC50 was calculated as 3.049 mg/L (95% CI -0.96,7.06) based upon the mean measured diethylene glycol dibenzate concentrations. The original study report only provided endpoint results based upon nominal loading rates. Subsequent to the study the EC50 endpoint was recalculated based upon the measured DEGDB concentrations that were provided in the study report. In the calculation of the EC50 the Chi-squared test was significant for a lack of fit using Probit, therefore the EC50 was calculated with the Trimmed Spearman Karber method using the TOXSTAT (v3.4) statistical software.

TABLE 1
Cumulative immobilisation data for Daphnia magna exposed for 48 hours to Benzoflex 2-45
Nominal initial loading rate mg/L	Cumulative immobilised Daphnia magna (initial population: 5 per replicate)
	24 hours						48 hours
	R1	R2	R3	R4	Total	%	R1	R2	R3	R4	Total %
Control	0	0	0	0	0	0	1	1	0	0	2	10
1.0	0	0	0	0	0	0	0	1	1	0	2	10
2.2	0	0	0	0	0	0	3	2	1	2	8	40
4.6	0	0	0	0	0	0	4	3	1	2	10	50
10	0	1	2	1	4	20	3	3	4	4	14	70
22	0	1	1	2	4	20	5	3	4	5	17	85
46	0	1	0	1	2	10	4	4	4	4	16	80
100	2	3	2	3	10	50	4	5	5	5	19	95

Immobilization Data @ 48 Hours Listed as Increasing Mean Measured Concentrations

Loading Rate (mg/L)	Measured Conc. (mg/L)	Number Immobilized (n=5)					Mean (%)
		R1	R2	R3	R4	Total
Control	ND	1	1	0	0	2	10
2.2*	0.23*	3	2	1	2	8	40
1*	0.31*	0	1	1	0	2	10
4.6	2.829	4	3	1	2	10	50
10	2.857	3	3	4	4	14	70
46*	3.521*	4	4	4	4	16	80
22*	13.305*	5	3	4	5	17	85
100	17.755	4	5	5	5	19	95
*Revised order to monotonically increasing mean measured concentrations for statistical analyses

Table 2
Benzoflex 2-45: Environmental parameters Mean values for pH, temperature and dissolved oxygen
Nominal initial loading rate mg/L	Mean values
	pH	mgO2/L	T°C
Control	7.8	9.0	19.3
1.0	7.8	8.5	19.3
2.2	7.8	8.6	19.3
4.6	7.7	8.3	19.3
10	7.7	8.3	19.3
22	7.7	8.2	19.3
46	7.7	8.1	19.3
100	7.7	8.1	19.3

Table 3: Post-Study Calculated DEGDB (dibenzoate) and DEGMB

(monobenzoate) Mean Measured Concentrations

Dibenzoate						Monobenzoate
Loading Rate (mg/L)	Initial Conc (mg/L)	Final Conc (mg/L)	Mean Conc (mg/L)			Loading Rate (mg/L)	Initial Conc (mg/L)	Final Conc (mg/L)	Mean Conc (mg/L)
Control	ND	ND	ND			Control	ND	ND	ND
1	0.5423	0.0873	0.315			1	0.08535	0.01857	0.05196
2.2	0.3928	0.0591	0.226			2.2	0.05778	0.0003*	0.02904
4.6	3.502	2.156	2.829			4.6	0.5423	1.214	0.87815
10	3.707	2.006	2.857			10	0.9448	1.429	1.1869
22	13.21	13.4	13.305			22	2.175	4.4	3.2875
46	4.267	2.774	3.521			46	3.779	4.431	4.105
100	17.79	17.72	17.755			100	8.884	8.657	8.7705
						*ND entered as half LOD (0.0006 mg/L) for mean

Validity criteria fulfilled:

yes

Conclusions:

The 48 hour EC50 (immobilisation value for DEGDB with Daphnia Magna) was determined to be 3.049 mg/L (95% CI -0.96,7.06) based upon the mean measured diethlene glycol dibenzoate concentrations.

Executive summary:

An acute Daphnia Immobilisation study was performed to determine the acute toxicity to Daphnia Magna of the test material diethylene glycol dibenzoate (DEGDB). The study was conducted according to EC, OECD, and US EPA test guidelines, and in compliance with GLP. The original study report reported the endpoint results based upon nominal loading rates. Subsequent to the study the EC50 endpoint was recalculated based upon the measured concentrations that were provided in the study report. The 48 hour EC50 (immobilisation value for DEGDB with Daphnia Magna) was determined to be 3.049 mg/L (95% CI -0.96,7.06) based upon the mean measured concentrations.

Description of key information

DEGDB:

Short-term toxicity to aquatic invertebrates:

- Key study, reliability 1, OECD 202, Daphnia magna - EC50 = 3.05 mg/L

 

DPGDB:

Short-term toxicity to aquatic invertebrates:

- Key study, reliability 1, OECD 202, Daphnia magna - EC50 = 5.0 mg/L

 

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

LC50

Effect concentration:

3.05 mg/L

Marine water invertebrates

Marine water invertebrates

Dose descriptor:

LC50

Effect concentration:

3.05 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 acute toxicity studies were carried out on Daphnia magna.  These data demonstrate that the components of the reaction mass have similar environmental profiles with all EC50 values in the same order of magnitude, >1.0 mg/L and <10 mg/L. The most sensitive endpoint result obtained for these components has been used as the key value to evaluate hazard and is highlighted below.

The key value is:

Aquatic toxicity to aquatic invertebrates EC50 = 3.05 mg/L (DEGDB)