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Short-term toxicity to aquatic invertebrates

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Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Toxicity test initiated February 20, 2018, and terminated February 22, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to other study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Principles of method if other than guideline:
The O-ring test system for preparing test solutions adhered to guidelines outlined in the OECD Series on Testing and Assessment Number 29 (OECD, 2001). General practices for testing of difficult test substances as outlined in OECD Series of Testing and Assessment Number 23 (OECD, 2000) were also incorporated as applicable.
The O-ring delivery system and analytical methods refined during multiple range finding trials provided a stable and precise exposure regime for conducting the toxicity trial.
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Dose verification samples of the test concentrations in test media were taken at test initiation, 24 hours of exposure, and termination (48 hours) in all control and test exposures. Five milliliter aliquots of each control and test concentration were withdrawn from the middle of the test vessel
and transferred to a vial containing 5 ml ethyl acetate. The samples were withdrawn with minimal agitation of the test vessel to ensure the test substance would not form micelles or films (vigorous mixing would potentially re-distribute test material that was not actually present as dissolved
material). Once the water samples were added, the sample vials were screwed closed and secured with electrical tape before shipment to Elevance.
Vehicle:
yes
Remarks:
methanol
Details on test solutions:
The 9-DAME was provided as solutions of substance in methanol, which contain silicone rubber passive loading devices.
The test substance (9-DAME) was loaded into the test vessels using a passive loading device patterned after studies reported by Butler et al. (2013), Smith et al. (2010), and Stibany et al. (2014). The passive loading device consisted of silicon rubber O-rings soaked in methanol, or methanol with 5 percent 9-DAME. The 9-DAME test vessel doses were generated by increasing the number of loaded 0-rings per vessel. Prior investigation indicated that 9-DAME would adhere to glass or plastic test vessel walls, and Teflon (PTFE) beakers would be required to stabilize soluble 9-DAME concentrations in the water column.

The O-rings were purchased from McMaster-Carr (1" ID, 1/16" width, ~0.30g/0-ring). Elevance prepared the O-rings loaded in a mixture of 5% test 9-DAME:
95% methanol, and O-rings soaked in 100% methanol for the solvent controls. The silicone O-rings are prepared with sequentially cleaning using ethyl acetate and methanol. The O-rings are dried in a 70 °C oven to ensure that all methyl esters and solvents are removed from the O-rings. The O-rings are then soaked in a percent level solution (5% test 9-DAME: 95% methanol for this protocol) of methyl ester in methanol for a minimum of 72 hours. The solutions (containing the silicone rubber O-rings) were delivered to Ramboll. The solutions containing 0-rings were stored at room temperature until prepared for toxicity testing.

The test system was dosed 24 hours prior to adding test organisms, by placing one or more preloaded silicon rubber O-rings in the test water. The O-rings were removed from the methanol solution and dried before placing them in the test containers with test medium. To dry, the O-rings were removed from solution using forceps and placed on clean dry paper towel, blotted with a separate dry paper towel to remove any remaining liquid on the surface, and then allowed to dry at room temperature for 30 minutes. The post blotting air drying time was investigated in previous range-finding tests to ensure excess solvent would not cause toxicity and still ensure the integrity of water column 9-DAME concentrations. O-rings were then transferred using forceps to the appropriate test containers 24 hours prior to adding test organisms to equilibrate the test solutions.
Test organisms (species):
Daphnia magna
Details on test organisms:
The test species was Daphnia magna, a commonly tested freshwater cladoceran, and was selected for testing because it is an ecologically important pelagic crustacean. In addition to its importance in food-web transfer of energy and nutrients, D. magna has long been used as a standard toxicity test organism, and is one of the Daphnia species identified as a suitable test organism in OECD Method 202. D. magna were obtained from in-house cultures maintained at Ramboll's test facilities in Brentwood, Tennessee. D. magna are continuously cultured in the United States Environmental Protection Agency (USEPA) reconstituted moderately hard water that served as the 0-rings test preparation and toxicity test medium in this study.

Culture conditions were essentially identical to the toxicity test conditions in terms of temperature, culture water, and photoperiod and light intensity. Information related to test organism health while in culture was documented and included notations regarding parent-organism mortality, absence of ephippia, and numbers of neonates produced. Feeding of adult D. magna while in culture, and feeding of neonates immediately prior test initiation (minimum of two hours prior to test initiation) consisted of feeding a green algae (Pseudokirchneriella subcapitata) and Cerophyll suspension (combined total solids concentration of 1,700 to 1,900 mg/L) at rates of 60 ml/Lin culture, and 17 ml/L immediately prior to neonate testing . Consistent with OECD Guideline 202, D. magna neonates less than 24 hours old were used to initiate toxicity tests, and were not fed during the test.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
84.0 mg CaCO3/L
Test temperature:
24.2 to 24.6 °C
pH:
7.82 s.u. to 7.97 s.u.
Dissolved oxygen:
6.5 to 8.5 mg/L
Conductivity:
221 to 247 μS/cm
Nominal and measured concentrations:
Nominal: Concentration dependent on number of O-rings used.
Measured:
T = 0 to 48 hr. Average 9-DAME Concentration
1 O-ring: 0.56 mg/L (min / max 0.40 / 0.73 mg/L)
2 O-rings: 0.88 mg/L (min / max 0.77 / 1.03 mg/L)
3 O-rings: 1.06 mg/L (min / max 0.71 / 1.27 mg/L)
4 O-rings: 1.41 mg/L (min / max 1.06 / 1.65 mg/L)
5 O-rings: 1.70 mg/L (min / max 1.35 / 2.11 mg/L)
Details on test conditions:
total alkalinity was 42.0 mg/L. Light intensity was 844 lux, and total residual chlorine (TRC) was below levels of detection. Photoperiod was 16-hr light: 8-hr dark.


Control and Dilution Water:
A reconstituted moderately hard laboratory water (USEPA, 2002), served as the test media and primary control water. A secondary (solvent) control was employed to evaluate potential toxicity caused by the release of carrier solvent (methanol) released from O-rings. Five solvent only soaked O-rings were added to each test vessel for the solvent control exposure. The test water media was prepared from de-ionized water to which reagent-grade salts were added. This water is commonly used in the culture and testing of a variety of organisms (including D. magna), and is a suitable water for OECD Method 202. The test water is also routinely confirmed to be free of contaminants such as pesticides and heavy metals, and confirmed to contain total organic carbon (TOC) concentrations of less than 1 mg/L.

Range-Finding Toxicity Test
In previous GLP studies conducted by Harlan (2012) and ABC (2014) labs, test concentrations were highly variable and measured concentrations declined rapidly in standard static-renewal and flowthrough tests. The 48-hr EC50, calculated using initial measured concentrations, was 1.7 mg/L 9 -DAME. However, measurements after 24 hours were mostly non-detect, so when a geometric mean concentration of initial and final measurements was calculated, the 48-hr EC50 was 0.8 mg/L. A 21 - day flow-through chronic test with D. magna found no effects to the Daphnia at a nominal loading rate of approximately 1.0 mg/L. Significant subsequent non-GLP work focused on obtaining stable test concentrations. Several types of passive loading devices, test container materials, loading rates and pre-test holding conditions were evaluated. Based on the earlier studies, and evaluation of O-rings as passive loading devices, a targeted exposure range bracketing 1 mg/L was selected.
A series of three "dry run" studies were conducted in 2017 to further refine the test parameters, and correlate the 0-ring delivery exposures with measured test concentrations. The sampling and analytical methods were also trialed in September 2017. Results from the dry run testing produced a dose regime centered on an approximate test concentration of 1 mg/L with sufficient replication to provide requisite precision.

Toxicity Test Conditions:
The dose regime was developed by preparing solutions with a definitive range of test substance concentrations. The 9-DAME test substance was delivered indirectly into the test media by placing 9-DAME loaded 0-rings into the test vessels of media.

Testing was conducted in a light- and temperature-controlled environmental chamber. Tests were conducted within the range of environmentally relevant and physiologically acceptable conditions for D.magna (i.e., pH 6.0 to 9.0 s.u., with sufficient dissolved oxygen concentrations to preclude non-toxicant related stress). The target test temperature was 25 +/- 2 °C, and the static, non-renewal test was conducted under a 16:8 L:D photoperiod. A light intensity of approximately 100 to 1,100 lux was maintained. Light intensity at the water surface was documented at test initiation and test termination. Test solution pH, specific conductance (conductivity), temperature, and dissolved oxygen were monitored at 24-hour intervals throughout the study. Test water residual chlorine, total hardness, and total alkalinity were documented at test initiation.

The test vessels were Teflon™ (PTFE) 250-ml beakers with 200 ml solution in each container. The test containers were loosely covered to reduce the loss of test solution or dilution water due to evaporation and to minimize the entry of dust or other particles into the solutions. All exposures consisted of four individual replicates for determination of toxicity test endpoints, and a surrogate replicate for assessment of water quality conditions. Each test replicate contained five D. magna neonates (less than 24 hour old at test initiation). All test vessels were labeled to identify the 9 -DAME O-ring exposure, and identify individual test and surrogate vessels. All control and test exposures were established using the same moderately hard water preparation.

Test organism immobility and water quality conditions were monitored and documented at 24-hour intervals. At test termination (48 +/- 1 hour), final water quality and D. magna immobility assessments were conducted and test solutions preserved for analyses. Any observations of unusual test organism activity (e.g., organisms floating or stuck to the bottom of the test vessel), stress, test material agglomeration in test vessels and on test organisms, or other test observations were also noted.

Reference substance (positive control):
yes
Remarks:
sodium chloride
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
1.27 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% confidence interval from 1.08 - 1.50 mg/L
Duration:
48 h
Dose descriptor:
other: EC25
Effect conc.:
1.01 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% confidence interval 0.98 to 1.09 mg/L
Duration:
48 h
Dose descriptor:
other: EC20
Effect conc.:
0.98 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% confidence interval 0.96 to 1.05 mg/L
Duration:
48 h
Dose descriptor:
EC10
Effect conc.:
0.93 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% confidence interval 0.70 to 1.02 mg/L
Details on results:
The experimental conditions utilized in this study maintained dissolved oxygen concentrations in the control exposures above 60 percent saturation (4.8 mg/Lat 25 °C), and maintained the pH of the test waters between 7.8 and 8.0 s.u. Thus, adequate test conditions were maintained.

Average control organism immobility was less than ten percent for control exposures. No signs of stress, floating organisms or entrapment on test vessel walls, or any other signs of unusual conditions were noted, in any exposures. All test acceptability criteria and other methodologies specified in the Study Protocol were met.

Biological Results:
The test acceptability criterion for the D. magna acute test was that there be no more than 10 percent of the control exposure daphnids trapped or immobilized at the water surface, and no more than 10 percent control organism immobility in 48 hours. For this test, there was no immobility of D. magna in either the primary water only control or the solvent control. Test organisms used in this testing were from cultures demonstrated to be within specified ranges of normal sensitivity based on sodium chloride reference toxicant testing.

The dose regime, based on increasing numbers of O-rings, demonstrated a graduated dose response with increasing dose causing increasing organism immobility. No immobility was observed for both 24 and 48-hour periods in the 1 O-ring and 2 O-ring exposures. Immobility rates at 24-hours increased from 20 percent in the 3 O-ring exposure to 50 percent in the 5 O -ring exposure. Immobility at 48-hours increased from 35 percent in the 3 O-ring exposure to 70 percent in the 5 O-ring exposure.
Complete (100 percent) immobility was not observed for any replicate of any 0-ring exposure.

The within exposure variability among replicates was low with the inter-replicate CV of 15.4 percent in the transitional 3 O-ring exposure group with 35 percent immobility effects. The CV was greater for the 4 O-ring exposure group (40.8 percent) with 60 percent of daphnia immobilized, and dropped to 38.5 percent for the 5 0-ring exposure group as percent immobilization increased to 70 percent. Comparing the range of the lower and upper 95 percent confidence intervals to the Trimmed Spearman-Karber EC50 value indicates that the 95 percent confidence interval is only 33 percent of the ECSO value.

Typically, test precision for the test may be expressed as Percent Minimum Significant Difference (PMSD) for a parametric No Observed Effect Concentration (NOEC) evaluation. However, in this case data assumptions for normality indicated a non-normal distribution of immobility data, and a nonparametric test (Steel Many-One Rank Sum Test) was conducted as the default statistic with a presumed test precision (PMSD) of 14.7 percent with the NOEC value defaulting to the lower 2 O-ring exposure for the NOEC. Evaluation of the individual replicate 9-DAME concentrations indicated overlap of measured concentrations between the different 0-ring exposures. As a result, the average 9-DAME concentration per group are not discreet, invalidating the premise of exclusive exposure regimes for valid NOEC determination.

Therefore, point estimates (ECx by linear interpolation) provide a valid estimate of effective dose at 10, 20, and 25 percent immobility effect, and are presented herein rather than the NOEC:
• EC10 of 0.93 mg/L (95% confidence interval 0.92 to 0.96 mg/L)
• EC20 of 0.98 mg/L (95% confidence interval 0.96 to 1.05 mg/L)
• EC25 of 1.01 mg/L (95% confidence interval 0.98 to 1.09 mg/L)

The confidence interval to ECx ratio is 4 percent for the EClO, 9 percent for the EC20 and 11 percent for the EC25. The review of confidence interval range to the ECx concentration indicates a high degree of precision in the dose-response data set.
Results with reference substance (positive control):
A reference toxicant test initiated February 21, 2018 with neonates from cultures used in this testing exhibited a sodium chloride 48 hour LCSO value of 3,693 mg/L NaCl which is within 3 percent of the running average of 3,783 mg/L for the previous 12 month period (database Coefficient of Variation, CV is 10 percent). Therefore, test organisms met TAC and cultures exhibited normal performance to the reference toxicant test.

Daphnia magna Immobilization Data:

Treatment Group

Rep ID

Initial Number Exposed

Number Immobilized (24 hr)

Total per concentration Immobilized (24 hr)

Percent Immobilized (24 hr)

Number Immobilized (48 hr)

Total per concentration immobilized (48 hr)

Percent immobilized (48 hr)

MH Water

A

5

0

0

0

0

0

0

B

5

0

0

C

5

0

0

D

5

0

0

Solvent Control (5 O-rings in Methanol)

A

5

0

0

0

0

0

0

B

5

0

0

C

5

0

0

D

5

0

0

1, O-ring

A

5

0

0

0

0

0

0

B

5

0

0

C

5

0

0

D

5

0

0

2, O-rings

A

5

0

0

0

0

0

0

B

4*

0

0

C

5

0

0

D

5

0

0

3, O-rings

A

5

0

4

20

2

7

35

B

5

2

2

C

5

2

2

D

5

0

1

4, O-rings

A

5

0

5

25

2

12

60

B

5

1

4

C

5

3

3

D

5

1

3

5, O-rings

A

5

1

10

50

3

14

70

B

5

3

3

C

5

4

4

D

5

2

4

 

Analytical Results

9-DAME Test Concentrations and Dose Verification:

The dose regime from one to five O-rings per replicate over the 48-hour test duration is well defined with minimal overlap except for the T=0 results for the 3 O-ring exposure. Average 9-DAME concentrations for each exposure group, and full test duration, range from 0.56 mg/L for the single O-ring replicates to 1.70 mg/L for the five O-ring exposures. Inter replicate variability was greatest for TO samples, with individual replicate measurements for the three O-ring exposure group range from 0.71 to 1.35 mg/L, and overlapping with values from the 1,

2, and 4, O-ring exposure groups. The CV for the 3 0-ring exposure at TO was elevated (CV = 28.1). Inter replicate variability and overlap between exposures was considerably reduced in the 3 0-ring exposure group at T24 and T48 durations.

Review of replicate concentrations from T0 to T48 samples indicates that the O-ring delivery system produced stable 9-DAME concentrations throughout the 48 hour test duration. The average (and standard deviation) 9-DAME concentration was calculated for each test replicate from T=0 to T=48 hours. The maximum CV for any individual test exposures was 21 percent (3 0-rings replicate D).

The CV values for all other test exposures ranged from 1 to 14 percent.

It should be noted that much of the test variability can be attributed to a single replicate in the 3 O-ring exposure group (replicate D). The variability and overlap between O-ring exposure groups precludes viable development of the NOEC endpoint.

Water Quality

The measured test and control water temperatures ranged from 24.2 to 24.6 °C, which is within the protocol-specified test range of 25°C and ± 1 °C. Dissolved oxygen measurements ranged from 6.5 to 8.5 mg/L, and pH ranged from 7.82 s.u. to 7.97 s.u. The total hardness value for the test water was 84.0 mg CaCO3/L, and total alkalinity was 42.0 mg/L upon test water preparation. The physical and chemical parameters for the toxicity test were the same conditions present for the D. magna cultures from which the toxicity test organisms were obtained. Light intensity was 844 lux, and total residual chlorine (TRC) was below levels of detection. Although the TRC detection limit of 0.02 mg/Lis somewhat higher than the desired limit per OECD Method 202, the test water was prepared from distilled water that is known to be free of contaminants, and was of acceptable quality for toxicity testing based on acceptable control organism response.

Measured concentrations

Experimental Design and Measured Concentrations

(Average measured 9-DAME concentration in four replicates)

9-DAME Loading Rate (O-rings per test vessel)

T=0 hr. Average 9-DAME Concentration (mg/L) (Min/Max)

T=24 hr. Average 9-DAME Concentration (mg/L) (Min/Max)

48 hr. Average 9-DAME Concentration (mg/L) (Min/Max)

T=0 to 48 hr. Average 9-DAME Concentration (mg/L) (Min/Max)

MH Water

<0.03

<0.03

<0.03

<0.03

Solvent control, 5 O-rings

<0.03

<0.03

<0.03

<0.03

1, 9-DAME O-rings

0.56 ( 0.42 / 0.73)

0.55 ( 0.42 / 0.70)

0.56 ( 0.40 / 0.71)

0.56 ( 0.40 / 0.73)

2, 9-DAME O-rings

0.88 (0.81 / 0.92)

0.89 (0.80 / 1.03)

0.89 (0.77 / 0.98)

0.88 (0.77 / 1.03)

3, 9-DAME O-rings

1.02 (0.71 / 1.35)

1.17 (1.08 / 1.27)

1.00 (0.94 / 1.08)

1.06 (0.71 / 1.27)

4, 9-DAME O-rings

1.38 (1.33 / 1.50)

1.48 (1.35 / 1.62)

1.35 (1.06 / 1.65)

1.41 (1.06 / 1.65)

5, 9-DAME O-rings

1.69 (1.35 / 1.83)

1.79 (1.58 / 2.11)

1.62 (1.53 / 1.77)

1.70 (1.35 / 2.11)

Validity criteria fulfilled:
yes
Conclusions:
The test substance (9-DAME) demonstrated acute toxicity to D. magna based on protocol conditions developed in accordance with OECD Method 202, and test exposure solutions prepared with a standardized O-ring delivery method. Analytical results indicated a well-defined dose regime with limited dose overlap between dosing groups (i.e. 1 thru 5, O-rings per test vessel) . The exposed D. magna demonstrated a dose response across the 9-DAME concentration gradient and an EC50 was calculated. The EC50 value, based on the measured average (both inter-replicate and complete 48 hour test duration) of 9-DAME concentrations in the water column was 1.27 mg/L 9-DAME (95% confidence interval from 1.08 - 1.50 mg/L).
Executive summary:

Test Substance: Methyl 9-Decenoate

Test Species: Daphnia magna

Test Duration: Test substance administration 1 day prior to loading organisms. Organism exposure to test substance duration 48-hours

T0-T48 Mean Measured Test Concentrations: 0 (water control), 0 (solvent control) 0.56, 0.88, 1.06, 1.41, and 1.70 mg/L 9-DAME

Nominal Test Concentrations: Test substance administered via 9-DAME loaded O-rings. 0 O-rings (water only), 5 solvent only O-rings (solvent control), 1 O-ring, 2 O-rings, 3 O- rings, 4 O-rings, 5 O-rings.

Results Summary:

Based on measured concentrations in test exposures (mg/L 9-DAME)

Test Endpoint

9-DAMEConcentration atTest Endpoint

 

48-hr. EC50

(95% Confidence Interval) Trimmed Spearman-Karber

 

1.27 (1.08 - 1.50)

48-hr. EC10

(95% Confidence Interval) LinearInterpolation(ICPIN)

 

0.90 (0.70 - 1.02)

48-hr. EC20

(95% Confidence Interval) LinearInterpolation(ICPIN)

 

0.98 (0 .96 - 1.05)

48-hr. EC25

(95% Confidence Interval) Linear Interpolation (ICPIN)

 

1.01 (0.98 - 1.09)

 

NOEC

 

Not Applicable*

 

*Not applicable due to overlap of measured concentrations in groups.

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Experimental Starting Date: 14 May 2012. Experimental Completion Date: 07 June 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study performed according to GLP and guideline
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
GLP compliance:
yes (incl. certificate)
Remarks:
Date of inspection: 19-21 July 2011. Date of signature: 31 Aug 2011
Analytical monitoring:
yes
Details on sampling:
Water samples were taken from the control and each surviving test group (replicates R1 –R4 pooled) at 0 (fresh media), 24 (old and fresh media) and 48 hours (old media) for quantitative analysis. Samples were stored at approximately -20 °C prior to analysis.

Duplicate samples were taken and stored at approximately -20 °C for further analysis if necessary.
Details on test solutions:
Saturated solutions were prepared by adding test material to Elendt M7 medium, stirring using a propeller stirrer at approximately 1500 rpm for 24 hours, followed by filtration of undissolved material through a 0.2 µm Gelman AcroCap filter to give 100% v/v saturated solution. Aliquots of the 100% v/v saturated solution were each separately diluted in final volumes Elendt M7 medium to give the lower dilutions for the tests.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Source: Derived from in-house cultures
- Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were first instar daphnids less than 24 hours old.
- Daphnids were not fed during the test
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Test temperature:
21°C to 22°C
Nominal and measured concentrations:
Nominal concentrations of 0.10, 1.0, 10 and 100 5 v/v saturated solutions for the range-finding test and 1.0, 1.8, 3.2, 5.6, 10, 18, 32, 56 and 100% v/v saturated solutions for the definitive test were prepared. The test preparations were observed to be clear, colourless solutions throughout the duration of the test.
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL glass jars containing approximately 200 mL of test preparation
- Aeration: No
- Renewal rate of test solution: For the test media renewal at 24 hours, the test concentrations were freshly prepared and the daphnids transferred by wide bore pipette from the 24-Hour old test media into the fresh test media.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4


TEST MEDIUM / WATER PARAMETERS
For the range-finding test reconstituted water was used and for the definitive test, Elendt M7 was used. The theoretical total water hardness of Elendt M7 medium and reconstituted water is 250 mg/L as CaCO3 therefore, in order to comply with the water characteristics for the OPPTS guideline, the prepared Elendt M7 or reconstituted water was diluted in a ratio of 50:50 with deionized reverse osmosis water prior to use to reduce the total water hardness


OTHER TEST CONDITIONS
- Photoperiod: 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods
- Light intensity: 927 to 962 lux.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Any immobilization or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia were considered to be immobilized if they were unable to swim for approximately 15 seconds after gentle agitation
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.8 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95% CL of of 0.68 – 0.94 mg/L
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
0.73 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.33 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
Range-finding test:
No immobilization was observed at the test concentrations of 0.10 and 1.0% v/v saturated solution. However, immobilization was observed at 10 and 100% v/v saturated solution (7 of 10 and 10 of 10, respectively). Based on this information test concentrations of 1.0, 1.8, 3.2, 5.6, 10, 18, 32, 56 and 100% v/v saturated solution were selected for the definitive test.

Chemical analysis of the test preparations at 0 hours showed measured concentrations to range from 0.163 to 11.6 mg/L. Analysis of the test preparations at 48 hours showed measured concentrations to range from 0.143 to 9.25 mg/L.

Definitive test:
Verification of Test Concentrations
Analysis of the 5.6 to 100% v/v saturated solution test concentrations at 0 hours showed measured concentrations to range from 0.832 to 19.7 mg/L. Analysis of the corresponding old media samples at 24 hours showed measured concentrations to range from less than the limit of quantitation (LOQ) of the analytical method (assessed as 0.087 mg/L) to 11.3 mg/L. At 24 hours the 32 to 100% v/v saturated solution test concentrations were not renewed due to 100% immobilization after 24 hours exposure. Analysis of the freshly prepared media for the 5.6 to 18% v/v saturated solution test concentrations at 24 hours showed measured concentrations to range from 0.717 to 2.78 mg/L whilst analysis of the corresponding old media at 48 hours showed measured concentrations to range from 0.178 to 0.519 mg/L.

Time-weighted mean measured test concentrations were calculated to be 0.33, 0.73, 1.5, 3.8, 7.6 and 15 mg/L.

Physico-chemical measurements:
Temperature was maintained at 21° C to 22 °C throughout the test, while there were no treatment related differences for oxygen concentration or pH.

The oxygen concentration in some of the test vessels was observed to have an air saturation value (ASV) in excess of 100%. This was considered to be due to the presence of microscopic air bubbles in the media super-saturating the diluent and was considered not to have had an impact on the outcome or integrity of the test as no adverse effects were observed in the control group.

Cumulative Immobilization Data in the Definitive Test:

Nominal Concentration (% v/v Saturated Solution)

Time-Weighted Mean Measured Concentration

(mg/L)

Cumulative Immobilized Daphnia
(Initial Population: 5 Per Replicate)

24 Hours

48 Hours

R1

R2

R3

R4

Total

%

R1

R2

R3

R4

Total

%

Control

Control

0

0

0

0

0

0

0

0

0

0

0

0

1.0

-

0

0

0

0

0

0

0

0

0

0

0

0

1.8

-

0

0

0

0

0

0

0

0

0

1*

1*

5*

3.2

-

0

0

0

0

0

0

0

0

0

0

0

0

5.6

0.33

0

0

0

0

0

0

0

0

0

0

0

0

10

0.73

1

2

0

2

5

25

1

2

2

2

7

35

18

1.5

4

4

3

3

14

70

5

5

5

5

20

100

32

3.8

5

5

5

5

20

100

5

5

5

5

20

100

56

7.6

5

5

5

5

20

100

5

5

5

5

20

100

100

15

5

5

5

5

20

100

5

5

5

5

20

100


R1– R4= Replicates 1 to 4

- Not applicable as no chemical analysis performed

* Considered to be due to natural causes rather than a toxic effect given that less than 10% immobilization occurred at this concentration and no immobilization was observed at the higher test concentrations of 3.2 and 5.6% v/v saturated solution.

Conclusions:
The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated. Given the decline in measured test concentrations it was considered justifiable to base the results on the time-weighted mean measured test concentrations.The 48-Hour EC50 value based on time-weighted mean measured test concentrations of 0.80 mg/L with 95% confidence limits of 0.68 – 0.94 mg/L. The Lowest Observed Effect Concentration was considered to be 0.73 mg/L. The No Observed Effect Concentration at 48 hours was 0.33 mg/L.

Description of key information

Key Study:

The objective of the study was to determine the acute toxicity of methyl 9 -Deceonoate to the freshwater invertebrate Daphnia magna through a static, 48 -hour non-renewal toxicity test.

The results of the study reported a 48-hour EC50 value of 1.3 mg/L (95% confidence interval of 1.1 to 1.5 mg/L). The study was conducted according to OECD Test Method 202 but used a passive dosing exposure modification to attain stable chemical exposures during the test. Previous acute ecotoxicity studies had identified issues with the stability of substance and declining concentrations.

The passive dosing was achieved by pre-soaking silicone O-rings in a methanol solution with 5% Methyl 9-decenoate, rinsing the O-rings in water and drying before placing one to five rings in PTFE test vessels containing the daphnids. Chemical analyses were conducted at times 0, 24 and 48 hours in each replicate system to document chemical concentrations.

No immobilizations were observed at the two lowest exposures (one O-ring, mean concentration of 0.56 mg/L and two O-rings, 0.88 mg/L). Mean percent immobilizations with three, four, and five O-rings were 35% (1.06 +0.18 mg/L), 60% (1.41 +0.17 mg/L) and 70% (1.70 +0.20 mg/L) respectively.

Chemical concentrations were stable throughout the 48 h test, with CV of 11.5 to 16.7 for the 3 highest exposures – thus meeting the OECD guidance of less than 20% variance during a test.

Support Study:

The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated. Given the decline in measured test concentrations it was considered justifiable to base the results on the time-weighted mean measured test concentrations. The 48-Hour EC50 value based on time-weighted mean measured test concentrations of 0.80 mg/L with 95% confidence limits of 0.68 – 0.94 mg/L.  The Lowest Observed Effect Concentration was considered to be 0.73 mg/L.  The No Observed Effect Concentration at 48 hours was 0.33 mg/L.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
1.27 mg/L

Additional information

A previous daphnid test has been reported as a supporting study, but is considered of limited reliability. Most significant, chemical concentrations declined by 43% to 95% over a 24 hr period. Consequently, exposures were estimated as the geometric mean of an initial measurement and a near-zero value (taken as half the Limit of Quantification). Further, initial concentrations in some exposures were measured as above the water solubility of methyl 9-decenoate, further limiting the credibility of the test results as representative of toxicity of a dissolved chemical.

The use of passive dosing with silicone O-rings has been reported in recent technical journals and is acknowledged in the most recent (draft) version of OECD No.23 Guidance document on Aqueous-phase Aquatic Toxicity Testing of Difficult Test Chemicals (August 2017). The study sponsor has extensively studied the use of silicone O-rings pre-loaded with methanol solutions of methyl 9-decanoate to provide a constant exposure. In addition, the use of PTFE vessels reduces the adsorption of material, minimizing the observed decrease in dissolved material over the course of a day. Based on the observed stability of test material in this daphnid study, it is considered as highly reliable and should be used instead of the previously reported study.