Juniper, Juniperus mexicana, ext., isomerized, acetylated

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10-06-2015 to 15-06-2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

An Acute Immobilisation Test to Daphnia magna STRAUS was carried out to determine the EL50-values of the Water Accommodated Fractions (WAF) of the test item after 24 and 48 hours of exposure under semi-static conditions in a closed system.

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

not applicable

Qualifier:

according to guideline

Guideline:

other: ECETOC (1996): Monograph No. 26, Aquatic Toxicity Testing of Sparingly Soluble, Volatile and Unstable Substances

Deviations:

not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: June 2013 ; signature: November 2013

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All loading levels and the control were analytically verified via GC-MS at the start (0 and 24 hours) and at the end of the exposure intervals (24 and 48 hours). Test item loadings were prepared as Water Accommodated Fraction (WAF). Definitive Test: Nominal concentrations: Five water accommodated fractions (WAF) were prepared with nominal loadings of the test item in the range: 0 (control), 0.563, 1.13, 2.25, 4.50, 9.00 mg/L WAF loading rates.
- Sampling method: At the start of the exposure intervals (0 and 24 hours), sampling was carried out after preparation of the test concentrations. At the end of the exposure intervals (24 and 48 hours), samples were taken directly from the test vessels. The method was validated prior to this study according to SANCO 3029/99 rev.4 (2000).
- Sample storage conditions before analysis: All original samples were stored at 6 ± 2 °C before preparation, if necessary. Prepared samples were stored in an autosampler at room temperature until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water-soluble test items and UVCB substances, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC, 1996 and OECD, 2000), is to expose the organisms to a Water Accommodated Fraction (WAF) of the test item in cases where the test item is a complex mixture and is poorly soluble in water and in the permitted solvents. Using this approach, aqueous media was prepared by mixing the test item with water for a prolonged period sufficient to ensure equilibration between the test item and the water phase. At the completion of mixing and following a settlement period, the test item phase was separated by siphon and the test organisms exposed to the aqueous phase, the WAF (which may contain dissolved and/or suspended and/or emulsified fractions of the test item mixture). Exposures were expressed in terms of the original concentration of the test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of the test item in the WAF. For each loading level, an appropriate amount of the test item (i.e. nominal 1.227, 2.463, 4.905, 9.810, 19.620 mg) was weighed out and transferred with 2180 mL of the dilution water (ISO test water) into a brown glass flask. With a content of 2180 mL, the brown glass bottle was nearly filled up and the headspace in the bottle was reduced to a minimum. These dispersions were shaken for 24 hours with 20 rpm at room temperature. After a separation phase of 24 hours, the aqueous phase or WAF was removed by side-arm of the flask (from the bottom of the glass flask). Five water accommodated fractions (WAF) were prepared with nominal loadings of the test item in the range: 0 (control), 0.563, 1.13, 2.25, 4.50, 9.00 mg/L WAF loading rates.
- Eluate: Not applicable.
- Differential loading: Not applicable.
- Controls: or positive control - reference item: 0.625, 1.0, 1.6 and 2.56 mg/L were prepared in a separately conducted reference test (documented in the full study report). A negative/blank control without test item or reference item was also included. Dilution water without test item tested under the same conditions as the test groups
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable.
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): Not applicable.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): None reported. Due to the density of the test item , any undissolved material was expected to float on the surface. The WAF's were checked via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion). No undissolved test item was present during preparation of the test concentrations (checked via Tyndall) and during the test (checked visually).
- Other relevant information: A semi-static test design with a daily renewal of the test solutions in glass flasks sealed with screw caps (made from polypropylene) under diffuse light conditions was chosen to reduce contact with air and losses of the test item by evaporation. The glass flasks were filled up with the test solutions having nearly no headspace.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphinds
- Strain: Daphnia magna STRAUS (Clone 5)
- Justification for species other than prescribed by test guideline: Not applicable.
- Age at study initiation (mean and range, SD): < 24 hours old daphnids from a healthy stock were used for the study
- Weight at study initiation (mean and range, SD): Not applicable.
- Length at study initiation (length definition, mean, range and SD): Not applicable.
- Stage and instar at study initiation: Juvenile ; < 24 hours
- Valve height at study initiation, for shell deposition study (mean and range, SD): Not applicable.
- Peripheral shell growth removed prior to test initiation: Not applicable.
- Method of breeding: Not reported. Although the breeder and culture conditions are given in the full study report.
- Source: in-house laboratory cultures
- Age of parental stock (mean and range, SD): Not applicable.
- Feeding during test: No. The daphnids were not fed during the study. During culture: The culture daphnids are fed at least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus, with an algae cell density of > 10^6 cells/mL. The algae are cultured at the test facility
- Food type: Not applicable.
- Amount: Not applicable.
- Frequency: Not applicable.

ACCLIMATION
- Acclimation period: At least 2 hours in dilution water.
- Acclimation conditions (same as test or not): Yes.
- Type and amount of food: Not applicable.
- Feeding frequency: Not applicable.
- Health during acclimation (any mortality observed): No.

QUARANTINE (wild caught)
- Duration: Not applicable.
- Health/mortality: Not applicable.

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES, INCLUDING CULTURING CONDITIONS: Not applicable.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

In accordance with the OECD TG 202 guideline.

Hardness:

Dilution water quality parameters: 0 hours: Total Hardness: 269 mg CaCO3/L ; 24 hours Total Hardness: 272 mg CaCO3/L

Test temperature:

Dilution water quality parameters: 0 hours: Temperature 20.4 °C ; 24 hours: Temperature 19.9 °C

pH:

Dilution water quality parameters: 0 hours: pH 7.97; 24 hours: pH 7.97; mean pH at 48 hours all replicates: did not differ by more than 1.5 units

Dissolved oxygen:

Dilution water quality parameters: 0 hours: O2 dissolved: 8.73 mg/L ; 24 hours: O2 dissolved: 9.73 mg/L; mean dissolved oxygen at 48 hours all replicates: did not decrease to less than 8.00 mg/L.

Nominal and measured concentrations:

Range finding tests were performed (non-GLP) prior to the definitive test: Information is provided in table 1. Measured concentrations were determined and are available in the full study report.
Definitive test: nominal concentrations:
Five water accommodated fractions (WAF) were prepared with nominal loadings of the test item in the range: 0 (control), 0.563, 1.13, 2.25, 4.50, 9.00 mg/L WAF loading rates.

Details on test conditions:

TEST SYSTEM
- Test vessel: 4.5 (ID) x 9.5 (H) cm, ca. 130 mL
- Type (delete if not applicable): closed airtight with reduced headspace; static.
- Material, size, headspace, fill volume: Sealed glass flasks with screw caps (made from polypropylene).
- Aeration: No aeration of the test solutions.
- No. of organisms per vessel: Control and test item: 20 per concentration, 5 per vessel (divided into 4 replicates)
- No. of vessels per concentration (replicates): 4 (four replicates, 5 daphnia per vessel).
- No. of vessels per control (replicates): 4 (four replicates, 5 daphnia per vessel).
- No. of vessels per vehicle control (replicates): Not applicable.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO Test water, according to OECD 202, Annex 3: composition (mg/L): KCl 5.75; NaHCO3: 64.8; CaCl2.2H2O: 294 ; MgSO4.7H2O: 123.
- Culture medium different from test medium: Yes. Elendt M4, according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L
The test vessels were filled up with the test solutions. The daphnids were inserted with a small amount of dilution water (start of the exposure) or test solution (water renewal) by pipette. Thereafter, the test vessels were closed immediately with screw caps. The test solutions were renewed after 24 hours. For this purpose, a second set of test vessels were filled with the freshly prepared test solutions and the daphnids were transferred by pipette.

OTHER TEST CONDITIONS
- Adjustment of pH: None. The test vessels were not pH adjusted during or after test item exposure.
- Photoperiod: 16/8 h light/dark cycle
- Light intensity: Diffuse light, light intensity of max. 20 µE /m2 s (1340 lx)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Immobility (including mortality), 24 hours and at 48 hours. An organism was considered to be immobile, if it was not able to swim in the water phase within 15 seconds after gentle agitation of the test vessel. Other observations were not made.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

3.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL: 2.66 - 3.66 mg/L; nominal based on WAF

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

1.15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL: 1.11 - 1.20 mg/L; nominal based on WAF

Details on results:

- Behavioural abnormalities: None
- Observations on body length and weight: Not applicable.
- Other biological observations: None.
- Mortality of control: No mortalities.
- Other adverse effects control: None.
- Immobilisation of control: None.
- Abnormal responses: None.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None. The WAFs were checked for a Tyndall effect which could not be observed. The tested solutions were visually clear throughout the exposure period.
- Effect concentrations exceeding solubility of substance in test medium: Not reported. Effect levels are based on loading rate (WAF).

Results with reference substance (positive control):

- Results with reference substance valid?: Yes.
- Mortality: None. Acute immobilisation observed only.
- EC50/LC50: 24h-EC50 was 2.00 mg/L (C.I. 1.76 - 2.27).
- Other: The EC50-value of the reference item potassium dichromate after 24 hours is within the prescribed concentration range of 0.6 - 2.4 mg/L of quality criteria according to AQS P 9/2 (02/2000) listed in DIN 38412 - L 30 for daphnids clone 5 cultured in Elendt M4 medium. The EC50-value of the reference item is also within the recommended range of 0.6 - 2.1 mg/L according to OECD Guideline TG 202

Reported statistics and error estimates:

EL50-values (after 24 and 48 hours) were estimated by sigmoidal dose response regression. The 95 % confidence limits were estimated from the standard error and the t-distribution. All calculations were carried out from the best-fit values with the software GraphPad Prism.
The loading-effect relationships are shown graphically. The EL50 values were given based on the nominal loading levels. Per definition of the WAF, all terms related to concentration levels have to be given as loading levels because partly dissolved compounds and mixtures cannot be related to concentrations. The EC50-value for the reference item and its 95 % confidence limits were calculated accordingly. All data were computer processed and rounded for presentation. Consequently, minor variations may occur from the original figures if manual calculations based on the original figures are made subsequently. Calculations were made using the following software: GraphPad Prism and MS Excel.

Table 1. Immobilisation Rates in the non GLP Preliminary Range Finding Test

(n = 20, divided into 2 replicates with 10 daphnids each)

Nominal loading level of the water accommodated fraction (WAF) [mg/L]	IMMOBILISATION [%]
	24 hours			48 hours
	Replicates			Replicates
	1	2	MV	1	2	MV
100	100*	100*	100	100*	100*	100
10	50*	50*	50	100*	100*	100
1	10	0	5	10	0	5
0.1	0	0	0	0	0	0
Control	0	0	0	0	0	0

* = Daphnids were dead and trapped on the surface of the water phase

** measured concentrations presented in the full study report

 

Table 2. Immobilisation Rates after 24 and 48 h of Exposure in the Definitive Test

(n = 20, divided into 4 replicates with 5 daphnids each)

Nominal loading of the test item [mg/L]	IMMOBILISATION [%]
	24 hours					48 hours
	Replicates					Replicates
	1	2	3	4	MV	1	2	3	4	MV
9.00	100	100	100	100	100	100	100	100	100	100
4.50	60	60	60	100	70	100	100	100	100	100
2.25	40	40	40	20	35	100	100	100	100	100
1.13	0	0	0	0	0	20	60	60	40	45
0.563	0	0	0	0	0	20	0	0	0	5
Control	0	0	0	0	0	0	0	0	0	0

** Per definition of the WAF, all terms related to concentration levels have to be given as loading levels because partly dissolved compounds and mixtures cannot be related to concentrations.

 

Table 3. Measured Concentrations and Percent of the Nominal and the Initially Measured Concentrations of the Test Item during the Definitive Test

Sampling date	0 hours Start of the exposure interval	24 hours End of the exposure interval		24 hours Start of the exposure interval	48 hours End of the exposure interval
Nominal loading of the test item [mg/L]	Test Item
	Meas. conc. [mg/L]	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	Meas. conc. [mg/L]	%
9.00	4.52	2.87	63	Not determined*
4.50	4.46	2.87	64	4.37	2.63	60
2.25	1.56 #1	1.34	86	1.41 #2	1.45	103
1.13	0.776 #1	0.739	95	0.964 #1	0.734	76
0.563	0.487	0.487	100	0.496	0.310	63
Control	< SysQL	< SysQL		< SysQL	< SysQL

Meas. conc. = measured concentration of the test item, dilution factors taken into account

% = percent of the initially measured concentration of the test item at 0 and 24 hours

SysQL = system quantification limit (1.00 µg test item/L)

#1 = mean value of two replicates, reanalysed on 2015-06-13

#2 = mean value of three replicates, 2 replicates reanalysed on 2015-06-13

* = Not determined, due to 100 % mortality after 24 hours

Validity criteria fulfilled:

yes

Conclusions:

The test item 48h-EL50 was 1.15 (95% CL: 1.11 – 1.20) mg/L based on nominal loadings into the WAF test system

Executive summary:

The acute toxicity to Daphnia magna was carried out according to OECD 202 Daphnia sp., Acute Immobilisation Test and EU Method C.2 guidelines under GLP. The study was conducted in a closed system (sealed glass flasks) without headspace under semi-static conditions over a period of 48 hours with five Water Accommodated Fractions (WAF) of the test item with loading levels in the range of 0.563 to 9.00 mg/L prepared in a geometric series with a separation factor of 2.0. The test item is a pale-yellow liquid and a UVCB substance. In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water-soluble test items and UVCB substances, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC, 1996 and OECD, 2000), is to expose the organisms to a Water Accommodated Fraction (WAF) of the test item in cases where the test item is a complex mixture and is poorly soluble in water and in the permitted solvents. Using this approach, the aqueous medium was prepared by stirring the test item with dilution water at 20 rpm for 24 hours at room temperature to ensure equilibration between the test item and the water phase. An appropriate volume of the test item was introduced into a brown glass flask filled with an appropriate amount of dilution water. The headspace in the glass flask was reduced to a minimum. After the completion of shaking and following a 24-hour settlement period, the aqueous phase was separated by siphon from the bottom of the brown glass flask. Due to the density of the test item, any undissolved material was expected to float on the surface. The WAFs were checked for a Tyndall effect via laser beam which could not be observed. The tested solutions were visually clear throughout the exposure period. The test organisms were exposed to the clear aqueous phase, the WAF. The test item was expected to evaporate. To reduce losses of the test item, the study was conducted in a closed system without headspace. Twenty daphnids were exposed to each loading level including an untreated control. The concentrations of the test item were analytically verified via GC-MS at the start (0 and 24 hours) and at the end of the exposure intervals (24 and 48 hours) in all concentration levels and the control. These analytical data were used as supporting information to monitor the stability of two constituents of the test item in the test system during the study. The measured concentrations of the test item were in the range of 63 to 100 % of the 0 hour measured concentrations at 24 hours and 60 to 103 % of the 24 hour fresh test solution measured concentrations at 48 hours. The water quality parameters (i.e. pH-value and dissolved oxygen concentration), measured at the start (0 and 24 hours) and at the end of the exposure intervals (24 and 48 hours), were within the acceptable limits. The validity criteria of the test guideline were fulfilled. The 48h-EL50 was 1.15 (C.I: 1.11 – 1.20) mg/L based on nominal loadings of the test item into the test system.

Description of key information

48h-EL50 (invertebrates) = 1.15 (C.I. 1.11 - 1.20) mg/L ; nominal concentratio based on WAF, 48hour-freshwater, OECD TG 202, 2015

Additional information

Key study : OECD TG 202, 2015 : The acute toxicity to Daphnia magna was carried out according to OECD 202 Daphnia sp., Acute Immobilisation Test and EU Method C.2 guidelines under GLP. The study was conducted in a closed system (sealed glass flasks) without headspace under semi-static conditions over a period of 48 hours with five Water Accommodated Fractions (WAF) of the test item with loading levels in the range of 0.563 to 9.00 mg/L prepared in a geometric series with a separation factor of 2.0. The test item is a pale-yellow liquid and a UVCB substance. In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water-soluble test items and UVCB substances, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC, 1996 and OECD, 2000), is to expose the organisms to a Water Accommodated Fraction (WAF) of the test item in cases where the test item is a complex mixture and is poorly soluble in water and in the permitted solvents. Using this approach, the aqueous medium was prepared by stirring the test item with dilution water at 20 rpm for 24 hours at room temperature to ensure equilibration between the test item and the water phase. An appropriate volume of the test item was introduced into a brown glass flask filled with an appropriate amount of dilution water. The headspace in the glass flask was reduced to a minimum. After the completion of shaking and following a 24-hour settlement period, the aqueous phase was separated by siphon from the bottom of the brown glass flask. Due to the density of the test item, any undissolved material was expected to float on the surface. The WAFs were checked for a Tyndall effect via laser beam which could not be observed. The tested solutions were visually clear throughout the exposure period. The test organisms were exposed to the clear aqueous phase, the WAF. The test item was expected to evaporate. To reduce losses of the test item, the study was conducted in a closed system without headspace. Twenty daphnids were exposed to each loading level including an untreated control. The concentrations of the test item were analytically verified via GC-MS at the start (0 and 24 hours) and at the end of the exposure intervals (24 and 48 hours) in all concentration levels and the control. These analytical data were used as supporting information to monitor the stability of two constituents of the test item in the test system during the study. The measured concentrations of the test item were in the range of 63 to 100 % of the 0 hour measured concentrations at 24 hours and 60 to 103 % of the 24 hour fresh test solution measured concentrations at 48 hours. The water quality parameters (i.e. pH-value and dissolved oxygen concentration), measured at the start (0 and 24 hours) and at the end of the exposure intervals (24 and 48 hours), were within the acceptable limits. The validity criteria of the test guideline were fulfilled. The 48h-EL50 was 1.15 (C.I: 1.11 – 1.20) mg/L based on nominal loadings of the test item into the test system.