Juniper, Juniperus mexicana, ext., isomerized, acetylated

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06-06-2015 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 201 (Alga, Growth Inhibition Test)

Version / remarks:

An Alga, growth inhibition test for determination of the EL10-, EL20- and EL50-values of growth rate and yield over a period of 72 hours. The study was carried out in closed bottles without headspace over a period of 72 hours in order to avoid losses of the test item to the headspace.

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

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

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/MS at the start (0 hours), after 24 and 48 hours and the end of exposure (72 hours). Test item loadings were prepared as Water Accommodated Fraction (WAF). Final test: Solutions containing: 0 (control), 0.316, 1.00, 3.16, 10.00, 31.60 and 100.00 mg/L prepared at a loading rate of WAF
- Sampling method: The concentration in all loading levels and the control were analytically verified via GC-MS/MS at the start (0 hours), after 24 and 48 hours and the end of exposure (72 hours). Separate replicates for the test item analysis at the beginning of the exposure were prepared without algae. For the test item analysis after 24, 48 and 72 hours, separate replicates were prepared with algae at the beginning of exposure and incubated under test conditions. 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, 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 were prepared by mixing the test item with water for a prolonged period sufficient to ensure equilibration between the test item and the water phase. Six water accommodated fractions (WAF) were prepared with nominal loadings of the test item in the range of 0.316 to 100 mg/L set up in a geometric series with a factor of: 0.316, 1.00, 3.16, 10.0, 31.6, 100 mg/L. For each loading level, an appropriate amount of the test item was weighed out and transferred into a brown glass flask with an appropriate amount of dilution water. The headspace in the brown glass flask was minimized by nearly filling up the flask. The dispersions were shaken for 24 hours with 20 rpm at room temperature. After a separation phase of 24 hours, the aqueous phases or WAFs were removed by side-arm of the flask (from the approximate bottom of the glass flask). Due to the density of the test item, any undissolved material is expected to float on the surface. The WAFs 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).
- Eluate: Not applicable.
- Differential loading: Not applicable.
- Controls: For positive control - reference item: potassium dichromate 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.
- 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.): Due to the density of the test item, any undissolved material is expected to float on the surface. The WAFs 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). Prior to start of the exposure, the test solutions were checked for undissolved test item. Presence of undissolved test item during preparation and during the test was not observed.
- Other relevant information: A static test was carried out. With regard to the volatility of the test item glass flasks without headspace were used to reduce losses of the test item.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: HINDÁK, SAG 61.81
- Source (laboratory, culture collection): Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen, Germany
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 35-70 µE·m-2·s-1 for 24 hours per day.

ACCLIMATION
- Acclimation period: No. However, a three days old preculture, prepared in dilution water, was used as inoculum.
- Culturing media and conditions (same as test or not): No. Culture Medium: Nutrient medium Z according to LÜTTGE et al. (1994) Botanica Acta, Journal of the German Botanical Society, No. 3 Volume 107 page 111-186 (June 1994), THIEME-VERLAG.
Dilution water: (mg/L) - NH4Cl: 15 ; MgCl2.6 H2O: 12 ; CaCl2.2 H2O: 18 ; MgSO4.7H2O: 15 ; KH2PO4: 1.6 ; FeCl3.6H2O: 0.064 ; Na2EDTA.2H2O: 0.1 ; H3BO3: 0.185 ; MnCl2.4H2O: 0.415 ; ZnCl2: 3x10-3 ; Na2MoO4.2H2O: 7x10-3 ; CoCl2.6H2O: 1.5x10-3 ; CuCl2.2H2O: 1x10-5 ; NaHCO3: 50 ; NaHCO3* : 250 ; MES monohydrate*: 2665.6 ; pH 8.1 +/- 0.2. This medium has a nominal hardness of 0.24 mmol Ca+Mg/L.
* additional compounds were added to enable sufficient growth under conditions without headspace.
- Any deformed or abnormal cells observed: None reported.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Remarks on exposure duration:

In accordance with the OECD TG 201 guideline.

Hardness:

Ca+Mg: 0.24 mmol/L

Test temperature:

Nominal range: 21 - 24 °C, controlled at ± 2°C - measured room temperature values were min: 22 ; max 23 and mean 22.5 °C

pH:

0 hours: pH 8.2 ± 0.2 ; 72 hours: pH 8.35-8.89 (definitive test concentrations) and pH 8.76 (controls). pH did not vary more than 1.5 units.

Nominal and measured concentrations:

Preliminary test: 0 (control), 1.0, 10 and 100 mg/L as a nominal loading of a WAF
Final test: Solutions containing: 0 (control), 0.316, 1.00, 3.16, 10.00, 31.60 and 100.00 mg/L prepared at a loading rate of WAF
See table 3 for nominal and measured concentrations at initial exposure and during the course of the test.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass.
- Type (delete if not applicable): Closed - Static.
- Material, size, headspace, fill volume: , volume: 119 mL, with aluminium tops with PFTE seals. Minimum headspace.
- Aeration: Vessel shaken continuously. Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm.
- Initial cells density: nominal: 5 x 10^3 - 10^4 and current: 5024 cells/ml
- Control end cells density: Mean (of replicates after 72 hours) 257553 cells/ml (or ca. x 51increase in cell density)
- No. of vessels per concentration (replicates): 3 replicates of each test concentration; Seperate replicates of each test group for sampling purposes
- No. of vessels per control (replicates): 6 replicates of the control
- No. of vessels per vehicle control (replicates): Not applicable.

GROWTH MEDIUM
- Standard medium used: Yes. adjusted-Medium M2. Additionally, compounds were added to enable sufficient growth under conditions without headspace. In accordance to OECD Guidance Document No. 23.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Prepared according to guidelines (see 'Details on test organisms' field for more details on composition).
- Culture medium different from test medium: Yes. (see 'Details on test organisms' field)
- Intervals of water quality measurement: Start and end of the test period.

OTHER TEST CONDITIONS
- Sterile test conditions: No.
- Adjustment of pH: No.
- Photoperiod: 24 hours ; continuous
- Light intensity and quality: 60-120 µE/m2/s ; within ± 15 % over incubation area
- Salinity (for marine algae): Not applicable.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal. No self-fluorescence was found at the concentration level of 100 mg/L in the range finding test.
- Other: Initial cell density: Microscopic evaluation of the cells was carried out daily. The cells were checked for any unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation and adherence of algae to test containers or aggregation of algae cells.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.16. In definitive test justified from the results of the range finding study.
- Justification for using less concentrations than requested by guideline: Not applicable.
- Range finding study: Yes.
- Test concentrations: Three replicates per concentration were exposed to dilutions representing 0.316, 1.0, 3.16, 10.0, 31.50 and 100 mg/L based on nominal loading in a WAF in a preliminary test. One extra test vessel per concentration without algae was used as background for the determination of the algal cell density at each time interval.
- Results used to determine the conditions for the definitive study: Yes.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

2.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 1.63 - 5.01 mg/L; nominal based on WAF

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

0.956 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CL: 0.843 - 1.05 mg/L; nominal based on WAF

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

0.492 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: < 0.316 - 0.822 mg/L; nominal based on WAF

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

0.439 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CL: 0.362 - 0.558 mg/L; nominal based on WAF

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

0.316 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: nominal based on WAF

Details on results:

- Exponential growth in the control (for algal test): Yes.
- Observation of abnormalities (for algal test): Microscopic evaluation of the cells at the start of the incubation period and at test end revealed no morphological abnormalities in the test item concentration or control.
- Unusual cell shape: No.
- Colour differences: None.
- Flocculation: Not reported.
- Adherence to test vessels:
- Aggregation of algal cells: No.
- Other:
- Any stimulation of growth found in any treatment: Yes. At low concentrations 0.316 mg/L nominal WAF loading a growth rate and yield inhibition was negative between at 72h. This low dose stimulation and was limited and was taken into account in the data analysis and effect level calculations.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Final test: During the exposure period the measured concentrations decreased in the range finder and definitive test from nominal values. No observations were made to account for the difference.
- Effect concentrations exceeding solubility of substance in test medium: Not reported. Effect concentrations based on loading rates (WAFs).

Results with reference substance (positive control):

- Results with reference substance valid?: Yes.
- EC50: The EC50 for growth rate reduction (ERC50: 0-72h) was 0.613 mg/L with a 95% confidence interval ranging from 0.589 to 0.638 mg/L with headspace. The EC50 for yield inhibition (EYC50: 0-72h) was 0.281 mg/L with a 95% confidence interval ranging from 0.234 to 0.314 mg/L.
The results with headspace and without headspace were within the test facility SOPs (historic values).
- Other: The sensitivity of the test system was in agreement with the historical data.

Reported statistics and error estimates:

The NOELR/LOELR were estimated by calculation of statistically significant differences of growth rate and yield inhibition. As a standard, One Way Analysis of Variance (ANOVA) and DUNNETT’s test were used for NOELR/LOELR calculations. When running a One Way Analysis of Variance, a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance tests are 0.05. The alpha-value (acceptable probability of incorrectly concluding that there is a difference) is alpha = 0.05. Normality Test failed in terms of growth rate. Therefore, a square data transformation was done to pass Normality Test.
Test for Normality
- Results Growth rate: Normality Test failed with untransformed data. Therefore, a square data transformation was performed. The differences in the mean values among the treatment groups are greater than would be expected by chance; there is a statistically significant difference (P = <0.001). Significant differences for the nominal loading rate of 1.00 mg/L of the test item and above compared to the control.
- Results for Yield: Normality (Passed; P = 0.707).The differences in the mean values among the treatment groups are greater than would be expected by chance; there is a statistically significant difference (P = <0.001). Significant differences for the nominal loading rate of 1.00 mg/L of the test item and above compared to the control.

Table 1. Results of the Preliminary Range Finding Test (non GLP, 0 - 72 hours)

Nominal loading level of the water accommodated fraction (WAF) [mg/L]	Growth Rate Inhibition   [%]	Yield Inhibition   [%]
100	30	78
10.0	22	67
1.00	8	33

Negative values = growth stimulation

Measured Exposure Concentrations during the Preliminary Test are presented in the full study report

 

Table 2. Percentage reduction in growth rate and inhibition of yield in the definitive test

Statistically significant differences of growth rates and yield compared to control values are marked (+), not significant differences are marked (-)

Nominal loading level of the water accommodated fraction (WAF)	Replicate	Growth Rate		Inhibition of Growth Rate	Yield		Inhibition of Yield
[mg/L]	No.	[d-1]		[%]	[cells/mL]		[%]
100	1		< LOQ	100		< LOQ	100
	2		-0.106	100		-1374	100
	3		< LOQ	100		< LOQ	100
	Mean	(+)	n.a.	100	(+)	n.a.	100
31.6	1		0.189	86		3829	98
	2		0.199	85		4108	98
	3		0.524	60		19148	92
	Mean	(+)	0.304	77	(+)	9028	96
10.0	1		0.414	68		12359	95
	2		0.408	69		12077	95
	3		0.405	69		11926	95
	Mean	(+)	0.409	69	(+)	12121	95
3.16	1		0.736	44		40740	84
	2		0.373	72		10349	96
	3		0.311	76		7737	97
	Mean	(+)	0.473	64	(+)	19609	92
1.00	1		1.09	17		126625	50
	2		1.07	19		118917	53
	3		1.05	20		113461	55
	Mean	(+)	1.07	18	(+)	119668	53
0.316			1.31	0		251300	0
			1.33	-2		269069	-7
			1.28	2		229260	9
	Mean	(-)	1.31	0	(-)	249876	1
Control	1		1.33			263863
	2		1.30			245467
	3		1.30			241811
	4		1.33			265563
	5		1.34			271879
	6		1.28			226589
	Mean		1.31			252529

negative inhibitions = increase of growth

n.a. = not determinable

< LOQ = below level of quantification

 

Table 3. Measured Concentrations and Percentage of the nominal concentration of the Test Item

Sampling date	0 h (Start of exposure)	24 h		48 h		72 h (End of exposure)
	Constituents 1 and 2 of the Test Item
Nominal loading level of the water accommodated fraction (WAF) [mg/L]	Meas. conc. [mg/L]	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%
100	3.49 #2	2.61	75	2.76	79	1.64	47
31.6	5.85	4.17	71	3.33	57	7.23 #3	123
10.0	5.16	3.97	77	5.28	102	4.45	86
3.16	1.95	2.02	103	1.74	89	1.18	61
1.00	0.826	0.771	93	0.770	93	0.622	75
0.316	0.193 #1	0.195	101	0.170	88	0.0976	51
Control	< SysQL	< SysQL		< SysQL		< SysQL

Meas. Conc = Measured concentration of the test item (dilution factor taken into account)

% = Percent of the initial measured concentration of the test item

SysQL = System quantification limit of the analytical method (0.001 mg/L test item)

#1 = Mean value of 3 replicates (reanalysed)

#2 = Mean value of 2 replicates (reanalysed)

#3 = Mean value of 2 replicates (reanalysed)

Validity criteria fulfilled:

yes

Conclusions:

The test item 72h-ErL50 (growth rate reduction) was 2.90 (C.I. 1.63 - 5.01) mg/L based on nominal loading in WAF. The corresponding ErL10 was 0.492(C.I. < 0.316 – 0.822) mg/L and the NOELR was 0.316 mg/L.

Executive summary:

The algal growth inhibition to Pseudokirchneriella subcapitata, was carried out according to OECD 201 Freshwater Alga and Cyanobacteria, Growth Inhibition Test and EU Method C.3 guidelines under GLP. The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours. The study was conducted under static conditions with an initial cell density of 5024 cells/mL. In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water-soluble test items, 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 were prepared by mixing the test item with water for a prolonged period sufficient to ensure equilibration between the test item and the water phase. Six Water Accommodated Fractions (WAF) were tested in a geometrical series with a dilution factor of √10 (nominal): 0.316, 1.00, 3.16, 10.0, 31.5 and 100 mg/L. For each loading level, an appropriate amount of the test item was weighed out and transferred into a brown glass flask with an appropriate amount of dilution water. The headspace in the brown glass flask was minimized by nearly filling up the flask. The dispersions were shaken for 24 hours with 20 rpm at room temperature. After a separation phase of 24 hours, the aqueous phases or WAFs were removed by side-arm of the flask (from the approximate bottom of the glass flask). Due to the density of the test item, any undissolved material is expected to float on the surface. The WAFs 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). Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits. The concentrations of the test item were analytically verified via GC-MS at the start of the exposure and after 24, 48 and 72 hours, respectively, in all test levels and the control. These analytical data were used as supporting information to monitor the stability of the three constituents of the test item in the test system during the study. The measured concentrations of the test item were in the range of 47 to 123 % of the 0 hour measured concentrations after 72 hours. All effect values given are based on nominal test item concentrations of the test item. 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. All the relevant validity criteria of the test guideline were fulfilled. The 72h-ErL50 (growth rate reduction) was 2.90 (C.I. 1.63 - 5.01) mg/L based on nominal loading in WAF. The corresponding ErL10 was 0.492(C.I. < 0.316 – 0.822) mg/L and the NOELR was 0.316 mg/L.

Description of key information

72h-ErL50 (algae; growth rate) = 2.90 (C.I. 1.63 - 5.01) mg/L ; WAF loading 72hour-freshwater, OECD TG 201, 2015

72h-ErL10 (algae; growth rate)= 0.492 mg/L (C.I. < 0.316 - 0.822 ) ; WAF loading 72 hour-freshwater, OECD TG 201, 2015

NOELR (algae; growth rate) = 0.316 mg/L ; WAF loading, 72 hour-freshwater, OECD TG 201, 2015

Additional information

Key study : OECD TG 201, 2015 : The algal growth inhibition to Pseudokirchneriella subcapitata, was carried out according to OECD 201 Freshwater Alga and Cyanobacteria, Growth Inhibition Test and EU Method C.3 guidelines under GLP. The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours. The study was conducted under static conditions with an initial cell density of 5024 cells/mL. In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water-soluble test items, 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 were prepared by mixing the test item with water for a prolonged period sufficient to ensure equilibration between the test item and the water phase. Six Water Accommodated Fractions (WAF) were tested in a geometrical series with a dilution factor of √10 (nominal): 0.316, 1.00, 3.16, 10.0, 31.5 and 100 mg/L. For each loading level, an appropriate amount of the test item was weighed out and transferred into a brown glass flask with an appropriate amount of dilution water. The headspace in the brown glass flask was minimized by nearly filling up the flask. The dispersions were shaken for 24 hours with 20 rpm at room temperature. After a separation phase of 24 hours, the aqueous phases or WAFs were removed by side-arm of the flask (from the approximate bottom of the glass flask). Due to the density of the test item, any undissolved material is expected to float on the surface. The WAFs 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). Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits. The concentrations of the test item were analytically verified via GC-MS at the start of the exposure and after 24, 48 and 72 hours, respectively, in all test levels and the control. These analytical data were used as supporting information to monitor the stability of the three constituents of the test item in the test system during the study. The measured concentrations of the test item were in the range of 47 to 123 % of the 0 hour measured concentrations after 72 hours. All effect values given are based on nominal test item concentrations of the test item. 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. All the relevant validity criteria of the test guideline were fulfilled. The 72h-ErL50 (growth rate reduction) was 2.90 (C.I. 1.63 - 5.01) mg/L based on nominal loading in WAF. The corresponding ErL10 was 0.492(C.I. < 0.316 – 0.822) mg/L and the NOELR was 0.316 mg/L.