Juniper, Juniperus oxycedrus, ext.

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:

04 October 2019 – 29 November 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0 (control), 1.2, 2.4, 5.1, 10.8, 47.6 and 100 mg/L.
- Sampling method: During Range finding study and main study, TOC was analyzed at 0 hour (Day 0) and at 72 hour (Day 3) from samples of all test nominal loading rates.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
The test item is a complex mixture (UVCB substance) which was found to be partially miscible (with no precipitation) in test medium (OECD Alga Medium) according to a lab internal method. Therefore, the Algae Test was carried out with aqueous extracts (WAF = Water Accommodated Fraction) at various loading rates of the test item in test medium.
In order to determine the optimum mixing duration to be used for WAF preparation, a water accommodated fraction (WAF) equilibration trail was performed prior to exposure: One individual WAF was prepared at each of three loading levels 1.0, 50.0 and 100 mg/L. At 2, 4, 6 and 24 hours after initiation of mixing, mixing was stopped at respective time points and the solutions were allowed to settle for one hour. A sample of WAF was removed from each loading level and mixing was resumed. The concentration of organic carbon that had solubilized into the WAF from the test item was measured using the TOC analyzer described above.
Maximum TOC values of 2.718, 25.57 and 51.98 mg/L was obtained at the nominal loading rate of 1.0, 50.0 and 100.0 mg/L after 6 hours of stirring followed by 1 hour of settling period, hence 6 hours of stirring duration, followed by 1 hour of settling period was selected for test media preparation.
Test media preparation: For every test item concentration, individual WAF was prepared by adding the appropriate amount of test item to the test medium. Each mixing vessel was filled to achieve minimal headspace given the constraints of the vessels and closed with foil-covered stoppers. The mixture was stirred for 6 h (based on equilibration trail) using a magnetic stirrer at minimum of 100 rpm. The mixtures were allowed to settle and equilibrate to test temperature (before removing the test solution). The aqueous portions of WAFs were collected using steri-pipette and transferred to test vessels.
- Controls: OECD Alga Medium only.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): The test substance was not fully soluble at the tested loading rate. However the method of preparing the WAFs was designed to mimimise exposure to undissolved test material.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green alga Pseudokirchneriella subcapitata
- Strain: ATCC 22662
- Source (laboratory, culture collection): American Type Culture Collection.
- Method of cultivation: Alga stock culture (maintained in OECD alga medium) is periodically subcultured at least once in a week and maintained with the illumination and temperature of 4440 to 8880 lux and 21 to 24°C, respectively. From healthy axenic culture of alga, pre-culture was prepared of desired cell density to provide the inoculum for test cultures.

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions (same as test or not): Same conditions as test. The algal cell density of pre-cultures (acclimatation period) for range finding study and main study was respectively 41850 and 42150 cells/mL at start.
- Any deformed or abnormal cells observed: Not reported.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None

Test temperature:

22.2ºC - 22.6ºC

pH:

7.95 - 8.18

Nominal and measured concentrations:

Nominal loading rates: 0 (control), 1.2, 2.4, 5.1, 10.8, 47.6 and 100 mg/L WAF

Details on test conditions:

TEST SYSTEM
- Test vessel: erlenmeyer flask
- Material, size, headspace, fill volume: glass, 250 mL capacity, 100 mL fill volume.
- Aeration: Not specified.
- Initial cells density: 5495 cells/mL
- Control end cells density: 936833 cells/mL (average)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): Not applicable

GROWTH MEDIUM
- Standard medium used: yes (OECD TG 201 medium)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: the OECD medium was prepared as per the OECD guideline 201.
- Culture medium different from test medium: No
- Intervals of water quality measurement: The temperature and light intensity was monitored once in a day during pre-culture and exposure. The pH of the test nominal loading rates was measured at the beginning (0 h) and at the end (72 h) of the exposure.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: No
- Photoperiod: Constant illumination
- Light intensity and quality: 5326 to 5935 lux was maintained with a universal white type fluorescent lamp

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations: counting chamber (observation times: 24, 48 and 72 hours): Cell density was obtained by calculating the average cell count (by summating the number of cells in all the four large corner squares and divide the sum by 4) and multiplied with chamber conversion factor (CCF) and dilution factor: Cell Density (cells/mL) = Average cell count × 10000 (CCF) × Dilution factor.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: geometric factor of 2.1
- Justification for using less concentrations than requested by guideline: Not applicable; 6 concentrations used for the main test.

- Range finding study
- Test concentrations: 0 (control), 0.01, 0.1, 1.0, 10.0, 50.0 and 100.0 mg/L.
- Results used to determine the conditions for the definitive study: At the tested concentrations of 0.01, 0.1, 1.0, 10.0, 50.0 and 100.0 mg/L, the inhibition growth rate was calculated to be 0.09, 0.59, 1.18, 8.24, 59.05 and 100% respectively.

Reference substance (positive control):

yes

Remarks:

(3, 5-Dichlorophenol )

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

26.68 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

(water-accommodated fraction loading rate)

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval = [20.56 – 34.61]

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

5.69 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

(water-accommodated fraction loading rate)

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval = [4.46 – 7.26]

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

7.04 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

(water-accommodated fraction loading rate)

Basis for effect:

biomass

Remarks on result:

other: 95% confidence interval = [5.36 – 9.24]

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

2.15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

(water-accommodated fraction loading rate)

Basis for effect:

biomass

Remarks on result:

other: 95% confidence interval = [1.45 – 3.19]

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

1.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

(water-accommodated fraction loading rate)

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

2.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

(water-accommodated fraction loading rate)

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): Not reported.

Results with reference substance (positive control):

- Results with reference substance valid? Yes, the 72 hours growth rate inhibition and percent inhibition in yield lie within the validity criteria acceptance range and establish the acceptability of test system response and test procedures followed.
- ErC50-72 h=1.12 mg/L and EyC50-72 h=0.33 mg/L.

Reported statistics and error estimates:

The 72 hours EL10, EL20 and EL50 values were calculated from the responses (growth rate and yield) data against log nominal loading rates using the Probit analysis method (Finney, 1971). NOELR and LOELR were determined by SPSS statistical procedure (t-test) for multiple sample comparison using individual replicate values of the specific growth rate.

Table 1: Mean cell density after exposure during range finding study

Group	Nominal Loading Rate  (mg/L)	R	Mean Cell Density (10^4Cells/mL)
			Observation Periods
			24 h	48 h	72 h
G1	0.0	R1	5.50	26.68	88.55
		R2	4.68	28.60	85.25
		R3	5.23	23.65	86.90
G2	0.01	R1	5.23	22.55	86.08
		R2	4.95	23.93	86.90
G3	0.1	R1	4.68	22.83	83.33
		R2	4.95	23.93	85.25
G4	1.0	R1	4.40	23.10	82.50
		R2	4.68	23.38	81.13
G5	10.0	R1	3.85	20.35	58.58
		R2	3.30	20.08	55.55
G6	50.0	R1	1.38	3.58	4.40
		R2	1.10	4.40	4.13
G7	100.0	R1	0.00	0.00	0.00
		R2	0.00	0.00	0.00

Table 2. Percent inhibition in cell density, growth rate and yield during range finding study

Group	Nominal Loading Rate (mg/L)	% inhibition in Yield	% inhibition in Cell Density	% inhibition in Growth Rate
G1	0.0	-	-	-
G2	0.01	0.48	0.47	0.09
G3	0.1	3.02	3.01	0.59
G4	1.0	5.89	5.85	1.18
G5	10.0	34.54	34.34	8.24
G6	50.0	95.65	95.09	59.05
G7	100.0	100.00	100.00	100.00

Table 3. Mean cell density after exposure during main study

Group	Nominal Loading Rate  (mg/L)	R	Mean Cell Density (10^4Cells/mL)
			Observation Periods
			24 h	48 h	72 h
G1	0.0	R1	6.33	25.58	96.53
		R2	6.05	26.40	92.95
		R3	5.78	27.23	93.23
		R4	6.33	22.55	94.05
		R5	5.23	23.93	92.68
		R6	6.05	26.40	92.68
G2	1.2	R1	5.50	26.40	93.78
		R2	6.33	29.15	93.23
		R3	5.78	25.58	91.03
G3	2.4	R1	4.68	22.55	83.33
		R2	4.40	20.90	82.78
		R3	5.23	21.45	78.65
G4	5.1	R1	3.03	14.58	48.13
		R2	3.03	15.13	48.13
		R3	2.48	14.85	41.53
G5	10.8	R1	2.48	9.63	21.45
		R2	1.93	9.08	22.00
		R3	2.48	9.90	21.45
G6	47.6	R1	1.10	2.75	4.13
		R2	1.38	2.20	5.23
		R3	1.10	1.93	3.85
G7	100.0	R1	0.00	0.00	0.00
		R2	0.00	0.00	0.00
		R3	0.00	0.00	0.00

Table 4. Average specific growth rate during main study

Group	Nominal Loading Rates (mg/L)	R	Growth Rate				Mean Growth Rate
			Observation Intervals (Days)
			0 to 1	1 to 2	2 to 3	0 to 3
G1	0	1	2.44	1.40	1.33	1.72	1.71
		2	2.40	1.47	1.26	1.71
		3	2.35	1.55	1.23	1.71
		4	2.44	1.27	1.43	1.71
		5	2.25	1.52	1.35	1.71
		6	2.40	1.47	1.26	1.71
		Mean	2.38	1.45	1.31	1.71
		±SD	0.07	0.10	0.07	0.01
		CV	3.02	6.98	5.72	0.30
G2	1.2	1	2.30	1.57	1.27	1.71	1.71
		2	2.44	1.53	1.16	1.71
		3	2.35	1.49	1.27	1.70
		Mean	2.37	1.53	1.23	1.71
		±SD	0.07	0.04	0.06	0.01
		CV	3.00	2.63	4.96	0.31
G3	2.4	1	2.14	1.57	1.31	1.67	*1.67
		2	2.08	1.56	1.38	1.67
		3	2.25	1.41	1.30	1.65
		Mean	2.16	1.51	1.33	1.67
		±SD	0.09	0.09	0.04	0.01
		CV	4.04	5.87	3.20	0.63
G4	5.1	1	1.71	1.57	1.19	1.49	*1.47
		2	1.71	1.61	1.16	1.49
		3	1.51	1.79	1.03	1.44
		Mean	1.64	1.66	1.13	1.47
		±SD	0.12	0.12	0.09	0.03
		CV	7.07	7.08	7.74	1.93
G5	10.8	1	1.51	1.36	0.80	1.22	*1.22
		2	1.25	1.55	0.89	1.23
		3	1.51	1.39	0.77	1.22
		Mean	1.42	1.43	0.82	1.22
		±SD	0.15	0.10	0.06	0.00
		CV	10.21	7.26	7.12	0.40
G6	47.6	1	0.69	0.92	0.41	0.67	*0.69
		2	0.92	0.47	0.87	0.75
		3	0.69	0.56	0.69	0.65
		Mean	0.77	0.65	0.65	0.69
		±SD	0.13	0.24	0.23	0.05
		CV	16.77	36.40	35.47	7.73
G7	100.0	1	0.0	0.0	0.0	0.0	*0.0
		2	0.0	0.0	0.0	0.0
		3	0.0	0.0	0.0	0.0
		Mean	0.0	0.0	0.0	0.0
		±SD	0.0	0.0	0.0	0.0
		CV	0.0	0.0	0.0	0.0

±SD: Standard deviation; CV: Coefficient of Variation; R: Replicate;*: Statistical significance of >0.05

Table 5. Yield of alga during main study

Group	Nominal Loading Rate (mg/L)	R	Cell density (Cells/mL)		Yield (72 hours)
			0 h	72 h
G1	0	1	5495	965250	959756
		2	5495	929500	924006
		3	5495	932250	926756
		4	5495	940500	935006
		5	5495	926750	921256
		6	5495	926750	921256
			Mean	936833	931339
			±SD	14826	14826
G2	1.2	1	5495	937750	932256
		2	5495	932250	926756
		3	5495	910250	904756
			Mean	926750	921256
			±SD	14552	14552
G3	2.4	1	5495	833250	827756
		2	5495	827750	822256
		3	5495	786500	781006
			Mean	815833	810339
			±SD	25552	25552
G4	5.1	1	5495	481250	475756
		2	5495	481250	475756
		3	5495	415250	409756
			Mean	459250	453756
			±SD	38105	38105
G5	10.8	1	5495	214500	209006
		2	5495	220000	214506
		3	5495	214500	209006
			Mean	216333	210839
			±SD	3175	3175
G6	47.6	1	5495	41250	35756
		2	5495	52250	46756
		3	5495	38500	33006
			Mean	44000	38506
			±SD	7276	7276
G7	100.0	1	5495	0	-5495
		2	5495	0	-5495
		3	5495	0	-5495
			Mean	0	0
			±SD	0	0

Table 6. Percent inhibition in cell density, growth rate and yield during main study

Group	Test Conc. (mg/L)	% inhibition in Yield	% inhibition in Cell Density	% inhibition in Growth Rate
G1	0	-	-	-
G2	1.2	1.1	1.1	0.2
G3	2.4	13.0	12.9	2.7
G4	5.1	51.3	51.0	13.9
G5	10.8	77.4	76.9	28.5
G6	47.6	95.9	95.3	59.7
G7	100.0	100.0	100.0	100.0

Table 7. Acceptance criteria for control during main study

0 – 72 Hours Mean Coefficient of Variation of Section-By-Section Average Specific Growth Rate
Daily average growth rate	±SD	% CV out of the daily growth rates
1.72	0.62	36.27
1.71	0.61	35.43
1.71	0.58	33.76
1.71	0.64	37.12
1.71	0.48	27.94
1.71	0.61	35.51
Mean % of CV 0 – 72 hours		34.34

Biomass increase during the whole test period
Initial biomass (cells/mL)	Cell density on day 3 (cells/mL)	Biomass increase Factor
5495	965250	175.68
5495	929500	169.17
5495	932250	169.67
5495	940500	171.17
5495	926750	168.67
5495	926750	168.67
Mean	936833.33	170.50

Table 8. TOC analysis during exposure

Dose Range Finding Study
Group	Nominal loading Rate (mg/L)	TOC Analysis in Test Media (mg/L)
		Day 0	Day 3
G1	0.0	-	-
G2	0.01	0.134	0.119
G3	0.1	0.782	0.647
G4	1.0	1.024	0.914
G5	10.0	8.691	9.695
G6	50.0	16.840	16.940
G7	100.0	31.260	31.300
Main Study
Group	Nominal loading Rate (mg/L)	TOC Analysis in Test Media (mg/L)
		Day 0	Day 3
G1	0.0	-	-
G2	1.2	1.084	0.935
G3	2.4	2.142	1.982
G4	5.1	3.118	3.176
G5	10.8	4.825	4.404
G6	47.6	28.960	21.410
G7	100.0	68.630	48.130

Table 9. Temperature and light intensity during pre-culture and exposure main study

Day	Temperature of the surrogate (°C)	Light intensity (Lux)				Revolutions per minute (rpm)
		1	2	3	4
During Acclimatisation (pre-culture) & Exposure
0	22.5	5841	5935	5629	5711	100
1	22.4	5489	5382	5921	5827	100
2	22.6	5654	5829	5713	5889	100
3	22.5	5826	5758	5656	5893	100
4	22.2	5744	5627	5758	5647	100
5	22.2	5465	5497	5692	5326	100
6	22.4	5682	5589	5642	5486	100
Mean	22.4	5672	5660	5716	5683	-
±SD	0.2	149.6	193.1	100.8	214.3	-
Min	22.2	5326
Max	22.6	5935

Table 10. pH of the test medium during main study

Group	Nominal Loading Rate (mg/L)	pH
		0 hours						72 hours
		R1	R2	R3	R4	R5	R6	R1	R2	R3	R4	R5	R6
G1	0	8.12	8.15	8.10	8.11	8.09	8.11	8.09	8.05	8.05	8.07	8.03	8.07
G2	1.5	8.10	8.17	8.18	-	-	-	8.05	8.06	8.10	-	-	-
G3	2.6	8.07	8.09	8.11	-	-	-	8.03	8.04	8.05	-	-	-
G4	4.8	8.08	8.09	8.09	-	-	-	8.00	8.05	8.01	-	-	-
G5	8.6	8.10	8.05	8.07	-	-	-	8.11	8.07	8.05	-	-	-
G6	15.4	8.07	8.10	8.11	-	-	-	8.00	8.01	8.01	-	-	-
G7	27.8	8.12	8.09	8.09	-	-	-	7.98	7.95	8.00	-	-	-
G8	100.0	8.09	8.06	8.1	-	-	-	8.09	8.05	8.05	8.07	8.03	8.07
	Min	8.05						7.95
	Max	8.18						8.11

Validity criteria fulfilled:

yes

Remarks:

(increase of biomass in control during 72h>16 fold; coefficient of variation of the mean specific growth rate among replicates in control (t0-t72)<7%; the mean of the replicate coefficients of variation in section-by-section growth rate in control < 35%)

Conclusions:

In an Alga Growth Inhibition Test with Pseudokirchneriella subcapitata green algae, the growth rate inhibition effect concentrations were determined to be 72 h-ErL50 = 26.68 mg/L and 72 h-ErL10 = 5.69 mg/L.

Executive summary:

An Algae Growth Inhibition Test was performed with Pseudokirchneriella subcapitata green algae on the test item over a period of 72 h in static conditions according to OECD Guideline 201, following GLP. The test item is a UVCB substance which was found to be partially miscible in OECD Alga Medium which was used as test medium. Therefore, the test was carried out with aqueous extracts (WAF = Water Accommodated Fraction) at various loading rates of the test item in the test medium. Based on a preliminary WAF equilibration trail, individual WAF solutions for every test item concentration were prepared in closed vessels with minimal headspace and stirred for 6 h. Then, the mixtures were allowed to settle and equilibrate to test temperature and aqueous portions of WAFs were transferred to the test vessels. In a preliminary range finding study the average growth inhibition rates were 0.09, 0.59, 1.18, 8.24, 59.05 and 100% at the tested nominal loading rates of 0.01, 0.1, 1.0, 10.0, 50.0 and 100.0 mg/L, respectively. Based on these results, the main test was conducted at nominal loading rates of 1.2, 2.4, 5.1, 10.8, 47.6 and 100 mg/L along with control group. The algal growth (cell density) was assessed at 24, 48 and 72 hours post-exposure. Exponentially growing alga cells (5495 cells/mL) were exposed to the selected nominal loading rates in 3 replicates for the treatment groups and 6 replicates for the control group. Negative control consisted of test medium alone and a solution of 3, 5-Dichlorophenol was used as reference substance. A growth rate inhibition of 0.2, 2.7, 13.9, 28.5, 59.7 and 100 % was observed at the nominal loading rates 1.2, 2.4, 5.1, 10.8, 47.6 and 100 mg/L respectively as compared with the control. Similarly, inhibition in yield of 1.1, 13.0, 51.3, 77.4, 95.9 and 100% was observed at the nominal loading rates 1.2, 2.4, 5.1, 10.8, 47.6 and 100 mg/L respectively as compared with the control. All validity criteria were fulfilled. TOC was analyzed at 0 hour (Day 0) and at 72 hour (Day 3) from samples of all test nominal loading rates. Based on these results, the growth rate inhibition effect concentrations were determined to be 72 h-ErL10 = 5.69 mg/L (95% CL: 4.46 – 7.26), 72 h-ErL20 = 9.67 mg/L (95% CL: 7.82 – 11.96) and 72 h-ErL50 = 26.68 mg/L (95% CL: 20.56 – 34.61), and the yield inhibition effect concentrations were 72h-EyL10 = 2.15 mg/L (95% CL: 1.45 – 3.19), 72h-EyL20 = 3.26 mg/L (95% CL: 2.36 – 4.42) and 72h-EyL50 = 7.04 mg/L (95% CL: 5.36 – 9.24). Finally, the 72 hours LOELR and NOELR were determined to be 2.4 and 1.2 mg/L respectively based on growth rate.

Description of key information

Key study. Test method according to OECD 201, GLP study. The growth rate inhibition effect concentrations in Pseudokirchneriella subcapitata green algae were determined to be 72 h-ErL50 = 26.68 mg/L (95% CL: 20.56 – 34.61) and 72 h-ErL10 = 5.69 mg/L (95% CL: 4.46 – 7.26).

Key value for chemical safety assessment

EC50 for freshwater algae:

26.68 mg/L

EC10 or NOEC for freshwater algae:

5.69 mg/L