Propane-1,2-diyl dibenzoate

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-12-11 to 2019-05-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

OECD 211, Daphnia magna Reproduction Test (OECD, 2012)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Name: Benzoflex 9-88 plasticizer
Synonym: Benzoflex 9-88
Batch/Lot No.: V988816101
CAS No.: Not Listed
Purity: 99.0%
Expiration Date: 31 October 2019
Date Received: 31 October 2018
Received from: Chemical Marketing Concept, New Milford, Connecticut

Analytical monitoring:

yes

Details on sampling:

Sampling Intervals and Solution Composition:
- Days 0, 7, 14, and 20 (new) Each treatment level and the control prior to division into replicate vessels
Days 1, 8, 15, and 21 (aged) Composite of all available replicate vessels within each treatment level and the control
Number of Samples Taken from Each Exposure Solution: One sample at each interval. Additional samples of the test solutions were also collected at each sampling interval and stored frozen as archive samples.
Sampling Location: Approximate midpoint from the surface, bottom, and sides of the vessel
Sampling Device: Pipet
QC Samples: Three samples per sampling interval, prepared in dilution water at nominal concentrations approximating the test concentration range

Water quality:
Total hardness, alkalinity, and conductivity were monitored at exposure initiation and weekly thereafter in the highest treatment level and the control during the exposure. On days 0, 7, and 14, these measurements were taken from freshly-prepared solutions. On day 21 (exposure termination), these measurements were taken from aged solutions. Total hardness and alkalinity of the exposure solutions were determined according to APHA et al. (2005).

Vehicle:

no

Details on test solutions:

Dilution Water During Testing

Water Type: Fortified laboratory well water
pH: 7.7 to 8.0
Dissolved Oxygen (mg/L): 8.5 to 10
Conductivity (µS/cm): 720 to 790
Total Hardness as CaCO3 (mg/L): 170 to 180
Total Alkalinity as CaCO3 (mg/L): 84 to 94
Total Organic Carbon: February 2019 = 0.95 mg/l; March 2019 = 0.64 mg/l.

Representative samples of the dilution water source were analyzed periodically for the presence of pesticides, PCBs, and toxic metals by Eurofins Lancaster Laboratories Environmental, Lancaster, Pennsylvania. None of these compounds have been detected at concentrations that are considered toxic in any of the water samples analyzed, in agreement with ASTM (2007) standard practice. Daphnid cultures are maintained in water from the same source as the dilution water utilized in this study and have successfully survived and reproduced over several generations. The acceptable performance of the daphnid cultures, in combination with the previously mentioned analyses, confirmed the acceptability of this dilution water for use during the conduct of bioassays.

Test Concentrations

Selection of nominal concentrations for the 21-day definitive exposure was based on the results of a stability/solubility trial, information developed at Smithers Viscient through a 17-day preliminary exposure.

Nominal Concentrations: Control, 2.6, 6.4, 16, 40, and 100% of 100 mg/L WAF

Co-Solvent: None

Primary Stock Solution Preparation

Primary Stock Concentration: 100 mg/L
Stock Solution Volume: 3.0 L
Stock Vessel: 4.0-L glass aspirator bottle
Amount of Test Substance Added (for example): 0.2996 g
Diluent: Fortified laboratory well water

The test substance was added directly to full volume of dilution water and was observed to be clear and colorless with oily globules of undissolved test substance on the surface. The solution was mixed slowly overnight using a Teflon-covered stir bar and magnetic stir plate. Following mixing overnight, the solution was observed to be clear and colorless with oily globules of undissolved test substance on the surface. The solubilized portion of the solution was carefully drained into a glass beaker from a spout on the bottom of the bottle, avoiding the surface of the solution. The resulting 100% of a 100 mg/L WAF solution was observed to be clear and colorless with no visible undissolved test substance following preparation. Based on stability information obtained during the trial, which determined that the stock was stable for up to 24 hours, the primary stock was prepared daily in the same manner as described above. The primary stock was used to prepare the exposure solutions at exposure initiation and at each renewal interval thereafter.

Test Solution Renewal

At exposure initiation and at each renewal interval (daily throughout the 21-day exposure with the exception of day 21), fresh Benzoflex 9-88 and control solutions were prepared. At each renewal period, freshly prepared solution was added to a second set of clean beakers until each vessel contained approximately 80 mL of solution. On renewal days, daphnids were carefully transferred from the aged solution into the freshly prepared exposure solutions. Addition of food suspensions occurred after daphnid transfer to freshly prepared solutions.

Test organisms (species):

Daphnia magna

Details on test organisms:

Test Organism
Daphnids were selected as the test organism since they are recommended by OECD and are commonly used in chronic freshwater invertebrate toxicity tests. The Daphnia magna used in this toxicity test were obtained from laboratory cultures maintained at Smithers Viscient. Daphnids were obtained for testing by removing all immature daphnids from the culture vessel, thus isolating sexually mature daphnids approximately 24 hours prior to exposure initiation. Young produced by these daphnids within 24 hours of isolation were used in the exposure. The culture water was prepared by fortifying well water based on the formula for hard water and sterilized by UV light and filtering it through an Amberlite XAD-7 resin column to remove any potential organic contaminants.

Test Organism Information
Species: Daphnia magna
Source: Smithers Viscient culture
Location: Wareham, Massachusetts
Age: <24 hours old at exposure initiation; immature daphnids were removed from culture <24 hours prior to exposure initiation, thus isolating sexually mature adults. Young produced by the parent organisms were used for the exposure.
Culture Vessel: 1.0-L glass beaker containing 0.80 L of water
Food Type: Unicellular green algae, Ankistrodesmus falcatus (4 × 10^7 cells/mL) and a suspension of YCT (yeast, cereal leaves, and flaked fish food)
Feeding Frequency: Once daily during culture
Ephippia Observed: No
Males Present: No

Culture Conditions Two Weeks Prior to Testing
Water Type: Fortified laboratory well water
pH: 7.5 to 8.0
Photoperiod: 16 hours light: 8 hours dark
Light Intensity: 49 to 75 footcandles (530 to 810 lux)
Temperature (°C): 19 to 21
Dissolved Oxygen (mg/L): 7.3 to 9.5
Conductivity (µS/cm): 750 to 790
Total Hardness as CaCO3 (mg/L): 180
Total Alkalinity as CaCO3 (mg/L): 90

Representative samples of the food source were periodically analyzed for the presence of pesticides, PCBs, and toxic metals by Eurofins Lancaster Laboratories Environmental, Lancaster, Pennsylvania. None of these compounds have been detected at concentrations considered toxic in any of the samples analyzed, in agreement with ASTM (2007) standard practice. Based on the analysis for pesticides, the food source was considered to be of acceptable quality since all analyte concentrations were below levels of concern.

Exposure solutions were mixed for approximately 1 minute using a glass rod. All exposure solutions were observed to be clear and colorless with no visible undissolved test substance following preparation. Freshly prepared solutions were divided into 10 replicate vessels, each labeled with the study number, treatment level/control, and replicate designation. Control vessels were maintained under the same conditions as the treatment level solutions. Exposure solutions were renewed daily. Freshly prepared exposure solutions for each treatment level and control were prepared following the procedures outlined above.

Biological Observations

The number of immobilized adult daphnids and observations of abnormal behavior were recorded daily. Immobilization is defined as the inability of daphnids to swim within 15 seconds of gentle agitation of the exposure vessel. The day of appearance of first brood in each exposure vessel was recorded. Live offspring were counted and removed daily beginning with the appearance of the first brood. The presence of aborted eggs and immobile offspring was recorded if observed and were removed. Observations of physical characteristics of exposure solutions (e.g., precipitate, cloudy solution) were recorded whenever the test organisms were observed.

At exposure termination (day 21), the total body length of each surviving adult daphnid was measured. Daphnids were measured (to the nearest 0.05 mm) from the apex of the head to the base of the shell spine using an Olympus SZH-ILLD-200 Stereoscope in combination with a calibrated ocular.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

180 mg/l

Test temperature:

20 +/- 1 °C

pH:

7.5-8.0

Dissolved oxygen:

7.3 to 9.5 mg/l

Conductivity:

750 to 790 µS/cm

Nominal and measured concentrations:

Nominal Concentrations: 2.6, 6.4, 16, 40, and 100% of 100 mg/L water accommodated fraction (WAF)
Geometric Mean Measured Concentrations: 0.12, 0.33, 0.83, 2.2, and 5.6 mg/L

Details on test conditions:

Exposure Initiation

The exposure was initiated when daphnids, <24 hours old, were impartially distributed to each of the 10 replicates for each nominal concentration and the control. Daphnids were impartially added to an intermediate beaker by adding no more than two daphnids to each beaker until all beakers contained two daphnids. This procedure was repeated until each intermediate beaker contained 10 daphnids. The exposure was initiated by transferring one daphnid to each exposure vessel from the intermediate beakers. The order of transfer was impartial for all treatment levels and the control.

Exposure Conditions

Temperature Controlled by: Environmental chamber set to maintain temperature at 20 ± 2 °C
Duration: 21 Days
Photoperiod: 16 hours light: 8 hours dark
Transition Period: 15- to 30-minute transition period was controlled with an automatic timer.
Light Intensity: 16 to 20 µE m-2-1 s measured with a Licor Model LI-250A photometer
Food Type: Algal suspension (Ankistrodesmus falcatus; 4 × 10^7 cells/mL): 250 µL (test days 0 through 7), 300 µL (test days 8 through 14), and 350 µL (test days 15 through 21).
Yeast, cereal leaves, and digested flaked fish food (YCT) suspension: 50 µL Equivalent to 0.1 mg carbon/daphnid/day
Feeding Frequency: Once daily
Replication: Ten replicates per treatment level and the control, each labeled to identify the study number, treatment level/control and replicate designation (A through J)
Control of Bias: Impartial addition of daphnids
Aeration: None
Exposure Vessel Size/Material: 100-mL clear glass beakers
Exposure Vessel Solution Volume: Approximately 80 mL
Exposure Vessel Cleaning: Cleaned prior to initiation

Key result

Duration:

21 d

Dose descriptor:

NOEC

Remarks:

reproduction

Effect conc.:

2.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

living offspring per surviving female

Duration:

21 d

Dose descriptor:

NOEC

Remarks:

immobilisation

Effect conc.:

5.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

immobilisation

Duration:

21 d

Dose descriptor:

NOEC

Remarks:

surviving adult length

Effect conc.:

2.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth

Duration:

21 d

Dose descriptor:

LOEC

Remarks:

reproduction

Effect conc.:

5.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

living offspring per surviving female

Duration:

21 d

Dose descriptor:

LOEC

Remarks:

immobilisation

Effect conc.:

> 5.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

immobilisation

Duration:

21 d

Dose descriptor:

LOEC

Remarks:

Surviving adult length

Effect conc.:

5.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth

Details on results:

Evaluation of Test Conditions

The results of water quality determinations made during the 21-day exposure period demonstrated that the dissolved oxygen concentration, temperature, pH, total hardness, total alkalinity, and conductivity of the exposure solutions were unaffected by the concentrations of test substance. These results established that the water quality conditions throughout the exposure remained within acceptable ranges for the survival, reproduction, and growth of Daphnia magna.

Analytical Results

Measured concentrations maintained the expected concentration gradient throughout the exposure with some decline in concentration observed over the 24-hour renewal interval. The most frequent renewal scheme (daily) was used for this exposure in order to maintain test concentrations as much as possible. Analysis of 23 of the 24 QC samples resulted in measured concentrations which were consistent with the predetermined recovery range (i.e., 70.0 to 120%) and ranged from 95.1 to 117% of the nominal fortified concentrations (0.00100, 1.00, and 10.0 mg/L for days 0 and 1 and 0.0150, 1.00, and 10.0 mg/L for all subsequent sampling intervals). Based on these results, it was demonstrated that satisfactory precision and quality control were maintained during the analysis of the exposure solutions.

The recovery of one QC sample on day 1 was 131% of the nominal concentration which is outside the acceptance criterion (i.e., 70.0 to 120%). Given the fact that the remaining QC samples for this interval are within the acceptable range, the failure of this QC sample to meet acceptance criteria has minimal impact on the evaluation of results for this testing.

Biological results

Dunnett’s Multiple Comparison Test determined a statistically significant effect in daphnid length for all concentrations, with the exception of the 16% of 100 mg/L WAF nominal concentration (0.83 mg/L mean measured concentration). When evaluating the data, the following points were considered:

- Comparison to effects observed in other endpoints - A significant effect was observed for reproduction at the 5.6 mg/L treatment level. Thus, the slight increase from approximately 1 to approximately 4% effect at 5.6 mg/L for length (compared to a flatter approximately 1% for the other concentrations) was considered to be toxicant related.
- Dose response – Since the four lower treatment levels were all similar percent effects (approximately 1%) and the high treatment level was slightly greater, this increase in effect seemed to be a dose response and toxicant related, which also corroborates the trend in other endpoints as mentioned above.
- Range-finding study corroboration - The preliminary exposure tested up to 100% of 100 mg/L WAF (the same nominal concentration as the high treatment for the definitive study). In the preliminary exposure, the daphnids in the high treatment were observed to be small during the exposure and produced no offspring. Based on the preliminary exposure, it appeared that the 100% of 100 mg/L WAF would be an effect concentration for length and reproduction. Statistical analysis of length from the preliminary exposure determined that this high concentration had a 17% effect compared to the control, a significant effect, after the 17-day exposure.
- Historical control data (HCD) – The daphnid length HCD mean is 4.77 ± 0.12 mm. The control mean for this exposure was 4.83 mm, which is within the first standard deviation for historical control data. All treatment level means fall within this first standard deviation (range = 4.66 - 4.90 mm) from the HCD mean, with the exception the high treatment level, which is just outside this range at 4.62 mm.

Considering this information, we believe it’s most appropriate to report the length NOEC and LOEC as 2.2 and 5.6 mg/L, respectively.

Based on nominal concentrations and the most sensitive endpoints, reproduction (total living offspring per surviving female) and length, the 21-day No-Observed-Effect Concentration (NOEC) and Lowest-Observed-Effect Concentration (LOEC) were determined to be 40 and 100% of 100 mg/L WAF, respectively (2.2 and 5.6 mg/L as geometric mean measured concentrations, respectively).

21-Day Chronic Exposure of Daphnids (Daphnia magna) to Benzoflex 9-88 – Statistical Analysis

Results Based on Nominal Concentrations (% of 100 mg/L WAF)

Endpoint	Survival	Reproduction (Total Living Offspring per Surviving Female)	Length
NOEC^a	100	40	40
LOEC^b	>100	100	100
EC10 (95% CI^c)	>100 (N/A^d)	45 (42-47)	>100 (N/A)
EC20 (95% CI)	>100 (N/A)	53 (50-56)	>100 (N/A)
EC50 (95% CI)	>100 (N/A)	86 (78-95)	>100 (N/A)

^aNOEC = No-Observed-Effect Concentration

^bLOEC = Lowest-Observed-Effect Concentration

^cCI = Confidence Interval

^dN/A = Not Applicable. EC value was empirically estimated; therefore, corresponding 95% confidence interval could not be determined

Results Based on Geometric Mean Measured Concentrations (mg/L)

Endpoint	Survival	Reproduction (Total Living Offspring per Surviving Female)	Length
NOEC^a	5.6	2.2	2.2
LOEC^b	>5.6	5.6	5.6
EC10 (95% CI^c)	>5.6 (N/A^d)	2.5 (2.3-2.7)	>5.6 (N/A)
EC20 (95% CI)	>5.6 (N/A)	3.0 (2.8-3.2	>5.6 (N/A)
EC50 (95% CI)	>5.6 (N/A)	4.9 (4.5-5.3)	>5.6 (N/A)

^aNOEC = No-Observed-Effect Concentration

^bLOEC = Lowest-Observed-Effect Concentration

^cCI = Confidence Interval

^dNA = Not Applicable. EC value was empirically estimated; therefore, corresponding 95% confidence interval could not be determined

Conclusions:

During a 21-day study, daphnia magna were exposed to dipropylene glycol dibenzoate to determined the chronic toxicity to aquatic invertebrates. A No Observed Adverse Concentration (NOEC) for reproduction and growth of 2.2 mg/l was determined.

Executive summary:

During a 21-day study, daphnia magna were exposed to dipropylene glycol dibenzoate to determined the chronic toxicity to aquatic invertebrates in a study performed in accordance with OECD TG 211. A No Observed Adverse Concentration (NOEC) for reproduction and growth of 2.2 mg/l was determined for the test item.

Description of key information

There are no key data available for propylene glycol dibenzoate (PGDB). Data available from a structural analogue Dipropylene glycol dibenzoate (DPGDB) has been used to read-across to PGDB to fill this requirement. The justification for read across is presented as an attachment included in Section 13 of the IUCLID dossier.

In a 21 -day Daphnia reproduction GLP study conducted according to OECD 211 a NOEC (reproduction and growth) of 2.2 mg/L (based on the measured geometric mean) was reported (Smithers Viscient, 2019; Klimisch score = 1).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

2.2 mg/L