Juniper, Juniperus oxycedrus, ext.

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:

18 October 2019 – 27 November 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0 (control), 2.6, 4.8, 8.6, 15.4, 27.8 and 50.0 mg/L (nominal loading rates)
- Sampling method: During Range finding study and main study, TOC was analyzed on day 0 (fresh) and on day 2 (spent) from samples for each loading rate.

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 (ISO reconstituted water) according to a lab internal method. Therefore, the Daphnia 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 1.855, 39.58 and 54.250 mg/L was obtained at the nominal loading rate of 1.0, 50.0 and 100.0 mg/L after 4 hours of stirring followed by 1 hour of settling period, hence 4 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 4 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: ISO reconstituted water 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):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
Age at study initiation (mean and range, SD): < 24 hours old.
- Source: Environmental and Molecular Toxicology Division, Department of Zoology, Karnataka University, Karnataka, India.
- Feeding during test: No

ACCLIMATION
- Acclimation period: 25 gravid female daphinds were acclimatized in reconstituted water for 48 hours prior to test (similar for range finding and main study). Offsprings produced on the first day were not used. Progenies produced on day two (less than 24 hours old) were transferred into the test containers.
- Acclimation conditions (same as test or not): same as test.
- Type and amount of food: live algal cells (Pseudokirchneriella subcapitata) at 2 mL per liter.
- Feeding frequency: at the beginning and on Day 2 of acclimatization.
- Health during acclimation (any mortality observed): Not specified.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

None

Hardness:

220 mg/L as CaCO3

Test temperature:

20.0 - 20.9ºC

pH:

7.52 - 7.98

Dissolved oxygen:

7.32 - 7.99 mg O2/L

Nominal and measured concentrations:

Nominal loading rates: 0 (control), 2.6, 4.8, 8.6, 15.4, 27.8 and 50.0 mg/L WAF

Details on test conditions:

TEST SYSTEM
- Test vessel: rectangular beaker
- Material, size, headspace, fill volume: glass, 100 mL (fill volume).
- Aeration: The dilution water was aerated prior to use for the test so that the dissolved oxygen concentration has reached saturation.
- Renewal rate of test solution (frequency/flow rate): 24 h
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): Not applicable
- Biomass loading rate: 20 mL of test solution per daphnid.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO reconstituted water prepared as described in the OECD 202 guideline.
- Intervals of water quality measurement: Total hardness was analyzed prior to its use for exposure. Temperature, pH and dissolved oxygen were recorded at test initiation and at the beginning and at the end of test in the treatments and in the control.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours of light and 08 hours dark cycle.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Each test vessel was observed for immobilized daphnids at 24 and 48 hours after the beginning of the test. Those animals not able to swim within 15 seconds after gentle agitation of the test beaker was considered to be immobile. In addition to immobility, any abnormal behavior or appearance was recorded.

VEHICLE CONTROL PERFORMED: Not applicable

RANGE-FINDING STUDY
- Test concentrations: 0.01, 0.1, 1.0, 10.0, 50.0 and 100.0 mg/L along with a control group.
- Results used to determine the conditions for the definitive study: no immobility was observed in control and at the nominal loading rate of 0.01, 0.1 and 1.0 mg/L; immobility of 20, 100 and 100% was observed at the nominal loading rates of 10.0, 50.0 and 100.0 mg/L during the 48 hours exposure period.

Reference substance (positive control):

yes

Remarks:

(Potassium dichromate)

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

14.95 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

(water-accommodated fraction loading rate)

Basis for effect:

mobility

Remarks on result:

other: 95% confidence limits: 13.34 - 16.77 mg/L.

Key result

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

4.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

(water-accommodated fraction loading rate)

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

LOELR

Effect conc.:

8.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

(water-accommodated fraction loading rate)

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities:
- Mortality of control: No mortality was observed over a period of 48 h in the control group.
- Abnormal responses: No clinical signs of toxicity were observed in control and at 2.6 mg/L during the 48-hour observation period. Clinical signs of localized on bottom of container and lethargy were observed at the nominal loading rates of 4.8, 8.6, 15.4 and 27.8 mg/L during the 48-hour observation period. Clinical signs of localized on bottom of container and lethargy were observed at the nominal loading rate of 50.0 mg/L during the 24-hour exposure period.

Results with reference substance (positive control):

- Results with reference substance valid? Yes, the 48 hour EC50 of the reference substance was within the acceptance range confirming the suitability of the test system. EC50 of Potassium dichromate on Daphnia magna is periodically determined. The date of the last study at the date of performing this test was 16-10-2019.
- EC50-48 h: 0.57 mg/L

Reported statistics and error estimates:

Data were analyzed by statistical method (Finney’s probit analysis) to calculate the slopes of the curves and the EL50 with confidence limits. One-way ANOVA was performed for 48 hour immobility for the determination of NOELR and LOELR.

Table 1. Summary of clinical signs and immobilisation during range finding study

Group	Nominal Loading Rate  (mg/L)	R	No. of Daphnids per replicate at start 0 h	Signs of toxicity and immobility of Daphnids Observed at
				24 h		48 h
				Toxic signs	I	Toxic signs	I
G1	0.0	R1	5	N(5)	0	N(5)	0
		R2	5	N(5)	0	N(5)	0
G2	0.01	R1	5	N(5)	0	N(5)	0
		R2	5	N(5)	0	N(5)	0
G3	0.10	R1	5	N(5)	0	N(5)	0
		R2	5	N(5)	0	N(5)	0
G4	1.0	R1	5	N(5)	0	N(5)	0
		R2	5	N(5)	0	N(5)	0
G5	10.0	R1	5	B(2), L(2)	1	B(2), L(2)	0
		R2	5	L(3), B(1)	1	L(3), B(1)	0
G6	50.0	R1	5	L(3)	2	-	3
		R2	5	B(2), L(2)	1	-	4
G7	100.0	R1	5	B(1)	4	-	1
		R2	5	B(1)	4	-	1

I: Immobilization; N: Normal; B: on Bottom (settled on the bottom of test vessel with just small movements); L: Lethargy; Letters outside and values inside the parentheses represent the clinical symptom code and number of Daphnids exhibiting that particular symptom, respectively.

Table 2. Summary of immobilisation at different time points of post exposure during range finding study

Group	Nominal Loading Rate  (mg/L)	R	Initial No. of Daphnids	Immobility (No.) of Daphnids		Cumulative No.		% of Immobility
				24 h	48 h	24 h	48 h	24 h	48 h
G1	0	R1	5	0	0	0	0	0	0
		R2	5	0	0
G2	0.01	R1	5	0	0	0	0	0	0
		R2	5	0	0
G3	0.1	R1	5	0	0	0	0	0	0
		R2	5	0	0
G4	1.0	R1	5	0	0	0	0	0	0
		R2	5	0	0
G5	10.0	R1	5	1	0	2	2	20	20
		R2	5	1	0
G6	50.0	R1	5	2	3	3	10	30	100
		R2	5	1	4
G7	100.0	R1	5	4	1	8	10	80	100
		R2	5	4	1

Table 3. Summary of clinical signs and immobilisation during main study

Group	Nominal Loading Rate  (mg/L)	R	No. of Daphnids per replicate at start 0 h	Signs of toxicity and immobility of Daphnids Observed at
				24 h		48 h
				Toxic signs	I	Toxic signs	I
G1	0	R1	5	N(5)	0	N(5)	0
		R2	5	N(5)	0	N(5)	0
		R3	5	N(5)	0	N(5)	0
		R4	5	N(5)	0	N(5)	0
G2	2.6	R1	5	N(5)	0	N(5)	0
		R2	5	N(5)	0	N(5)	0
		R3	5	N(5)	0	N(5)	0
		R4	5	N(5)	0	N(5)	0
G3	4.8	R1	5	N(4), B(1)	0	N(3), B(1)	1
		R2	5	N(3), B(2)	0	N(3), B(2)	0
		R3	5	N(3), B(1), L(1)	0	N(3), B(1), L(1)	0
		R4	5	N(3), L(2)	0	N(2), L(2)	1
G4	8.6	R1	5	N(2), B(1), L(1)	1	N(1), B(1), L(1)	1
		R2	5	N(2), L(2)	1	N(2), L(2)	0
		R3	5	N(3), L(2)	0	N(2), L(2)	1
		R4	5	N(3), L(2)	0	N(3), L(2)	0
G5	15.4	R1	5	N(2), L(1)	2	N(2), L(1)	0
		R2	5	N(2), B(1), L(1)	1	B(1), L(1)	2
		R3	5	N(1), B(2), L(1)	1	B(2), L(1)	1
		R4	5	B(2), L(1)	2	B(1), L(1)	1
G6	27.8	R1	5	B(2)	3	B(1)	1
		R2	5	B(2), L(1)	2	L(1)	2
		R3	5	B(3), L(1)	1	L(1)	3
		R4	5	B(2), L(1)	2	L(1)	2
G7	50.0	R1	5	L(2), B(1)	2	-	3
		R2	5	L(2)	3	-	2
		R3	5	B(1), L(1)	3	-	2
		R4	5	B(2)	3	-	2

I: Immobilization; N: Normal; B: on Bottom (settled on the bottom of test vessel with just small movements); L: Lethargy; Letters outside and values inside the parentheses represent the clinical symptom code and number of Daphnids exhibiting that particular symptom, respectively.

Table 4. Summary of immobilisation at different time points of post exposure during main study

Group	Nominal Loading Rate  (mg/L)	R	Initial No. of Daphnids	Immobility (No.) of Daphnids		Cumulative No.		% of Immobility
				24 h	48 h	24 h	48 h	24 h	48 h
G1	0	R1	5	0	0	0	0	0	0
		R2	5	0	0
		R3	5	0	0
		R4	5	0	0
G2	2.6	R1	5	0	0	0	0	0	0
		R2	5	0	0
		R3	5	0	0
		R4	5	0	0
G3	4.8	R1	5	0	1	0	2	0	10
		R2	5	0	0
		R3	5	0	0
		R4	5	0	1
*G4	8.6	R1	5	1	1	2	4	10	20
		R2	5	1	0
		R3	5	0	1
		R4	5	0	0
*G5	15.4	R1	5	2	0	6	10	30	50
		R2	5	1	2
		R3	5	1	1
		R4	5	2	1
*G6	27.8	R1	5	3	1	8	16	40	80
		R2	5	2	2
		R3	5	1	3
		R4	5	2	2
*G7	50.0	R1	5	2	3	11	20	55	100
		R2	5	3	2
		R3	5	3	2
		R4	5	3	2

*: Statistical significance of >0.05.

Table 5. TOC analysis during exposure

Dose Range Finding Study
Group	Nominal loading Rate (mg/L)	TOC Analysis in Test Media (mg/L)
		Day 0	Day 2
G1	0.0	0	0
G2	0.01	0.2772	1.094
G3	0.1	0.845	0.761
G4	1.0	1.423	1.123
G5	10.0	2.436	2.178
G6	50.0	21.680	18.800
G7	100.0	48.920	43.630
Main Study
Group	Nominal loading Rate (mg/L)	TOC Analysis in Test Media (mg/L)
		Day 0	Day 2
G1	0.0	0	0
G2	2.6	1.388	0.570
G3	4.8	1.330	0.352
G4	8.6	2.729	1.740
G5	15.4	5.908	4.177
G6	27.8	11.250	8.134
G7	50.0	27.370	20.670

Table 6. Physico-chemical parameters of test medium during range finding study

Group	Nominal Loading Rate (mg/L)	R	Dissolved Oxygen  (mg/L)		Temperature (°C)		pH
			0 h	48 h	0 h	48 h	0 h	48 h
G1	0.0	R1	7.80	7.70	20.9	20.2	7.59	7.48
		R2	7.90	7.74	21.0	20.3	7.62	7.50
G2	0.01	R1	7.91	7.78	20.7	20.1	7.63	7.51
		R2	7.90	7.75	20.9	20.0	7.65	7.54
G3	0.1	R1	7.85	7.71	20.6	19.9	7.62	7.54
		R2	7.87	7.74	20.9	20.1	7.60	7.54
G4	1.0	R1	7.83	7.64	21.1	19.9	7.57	7.5
		R2	7.81	7.62	21.0	19.8	7.52	7.45
G5	10.0	R1	7.82	7.60	20.9	20.2	7.54	7.42
		R2	7.84	7.63	20.8	20.3	7.56	7.41
G6	50.0	R1	7.80	7.54	20.7	20.1	7.50	7.43
		R2	7.78	7.57	20.8	20.0	7.45	7.40
G7	100.0	R1	7.78	7.58	20.9	20.2	7.45	7.38
		R2	7.75	7.50	20.9	20.2	7.47	7.37
Min			7.50		19.80		7.37
Max			7.91		21.10		7.65

Min: Minimum; Max: Maximum;

Table 7. Physico-chemical parameters of test medium during main study

Group	Nominal Loading Rate (mg/L)	R	Dissolved Oxygen  (mg/L)				Temperature (°C)				pH
			0 h	24 h	24 h	48 h	0 h	24 h	24 h	48 h	0 h	24 h	24 h	48 h
			F	S	F	S	F	S	F	S	F	S	F	S
G1	0	R1	7.95	7.81	7.82	7.80	20.9	20.4	20.5	20.2	7.87	7.79	7.81	7.77
		R2	7.90	7.86	7.82	7.76	20.9	20.5	20.8	20.2	7.89	7.72	7.83	7.79
		R3	7.96	7.91	7.84	7.81	20.8	20.9	20.7	20.0	7.98	7.84	7.80	7.75
		R4	7.95	7.75	7.86	7.81	20.9	20.4	20.4	20.3	7.85	7.73	7.85	7.78
G2	2.6	R1	7.91	7.84	7.82	7.75	20.9	20.5	20.5	20.1	7.80	7.71	7.79	7.65
		R2	7.92	7.83	7.75	7.70	20.7	20.6	20.6	20.4	7.85	7.76	7.90	7.69
		R3	7.93	7.81	7.83	7.78	20.7	20.9	20.4	20.2	7.85	7.73	7.81	7.63
		R4	7.99	7.93	7.83	7.80	20.8	20.7	20.5	20.2	7.82	7.7	7.78	7.68
G3	4.8	R1	7.87	7.83	7.83	7.80	20.6	20.5	20.7	20.0	7.85	7.62	7.73	7.64
		R2	7.85	7.79	7.73	7.68	20.5	20.7	20.9	20.0	7.8	7.73	7.70	7.63
		R3	7.8	7.78	7.78	7.65	20.7	20.6	20.5	20.1	7.81	7.73	7.68	7.59
		R4	7.87	7.82	7.80	7.67	20.8	20.4	20.4	20.0	7.85	7.74	7.72	7.6
G4	8.6	R1	7.85	7.79	7.73	7.63	20.8	20.9	20.7	20.4	7.76	7.68	7.69	7.62
		R2	7.8	7.74	7.71	7.60	20.5	20.5	20.6	20.2	7.70	7.65	7.73	7.59
		R3	7.75	7.74	7.78	7.62	20.9	20.4	20.5	20.1	7.75	7.69	7.72	7.63
		R4	7.70	7.66	7.81	7.65	20.6	20.3	20.5	20.2	7.72	7.64	7.79	7.63
G5	15.4	R1	7.80	7.73	7.65	7.57	20.9	20.4	20.4	20.3	7.74	7.62	7.72	7.6
		R2	7.81	7.72	7.73	7.68	20.6	20.4	20.8	20.1	7.75	7.71	7.98	7.96
		R3	7.74	7.68	7.68	7.60	20.7	20.5	20.4	20.4	7.71	7.67	7.68	7.58
		R4	7.72	7.62	7.73	7.66	20.8	20.6	20.7	20.3	7.77	7.63	7.62	7.59
G6	27.8	R1	7.58	7.45	7.62	7.51	20.7	20.9	20.6	20.5	7.76	7.71	7.70	7.61
		R2	7.50	7.38	7.58	7.49	20.4	20.7	20.4	20.3	7.78	7.65	7.69	7.64
		R3	7.67	7.41	7.53	7.45	20.5	20.5	20.5	20.1	7.79	7.63	7.72	7.65
		R4	7.65	7.52	7.55	7.47	20.9	20.7	20.7	20.7	7.77	7.71	7.79	7.7
G7	50.0	R1	7.55	7.46	7.62	7.50	20.7	20.5	20.5	20.4	7.62	7.59	7.62	7.55
		R2	7.51	7.38	7.53	7.41	20.8	20.4	20.4	20.1	7.65	7.61	7.62	7.57
		R3	7.48	7.32	7.43	7.38	20.7	20.6	20.5	20.0	7.58	7.52	7.67	7.55
		R4	7.57	7.43	7.50	7.43	20.5	20.5	20.8	20.1	7.61	7.54	7.69	7.54
Min			7.32				20.0				7.52
Max			7.99				20.9				7.98

Min: Minimum; Max: Maximum; F: Fresh; S: Spent

Validity criteria fulfilled:

yes

Remarks:

(Immobilisation in control < 10% (no immobilisation observed); dissolved oxygen concentration > 3 mg O2/L)

Conclusions:

In a short-term toxicity test to Daphnia magna, the 48h-EL50 of the test substance was found to be 14.95 mg/L.

Executive summary:

An acute aquatic toxicity study with Daphnia magna was conducted on test item according to OECD guideline 202, following GLP. The test item is a UVCB substance which was found to be partially miscible in ISO reconstituted water 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 4 h. Then, the mixtures were allowed to settle and equilibrate to test temperature and aqueous portions of WAFs were transferred to the test vessels. A range finding study was conducted for 48 h with nominal loading rates of 0.01, 0.1, 1.0, 10.0, 50.0 and 100.0 mg/L along with a control group. The immobilization percentages of 20, 100 and 100 % were observed at 10, 50 and 100 mg/L respectively. No immobilization was observed in control and at 0.01, 0.1 and 1.0 mg/L. Based on these results, the main test was conducted for 48 h at nominal loading rates of 2.6, 4.8, 8.6, 15.4, 27.8 and 50 mg/L along with control group. The test was conducted in semi-static conditions with 4 replicates of 5 daphnids per replicate. Negative control consisted of test medium alone and a solution of potassium dichromate was used as reference substance. No clinical signs of toxicity were observed in control and at 2.6 mg/L. Clinical signs of localized on bottom of container and lethargy were observed at 4.8, 8.6, 15.4 and 27.8 mg/L at 48h and at 50.0 mg/L at 24 h. The immobilization percentages of 10, 20, 50, 80 and 100 % were recorded at the tested nominal loading rates of 4.8, 8.6, 15.4, 27.8 and 50 mg/L during the 48 hour observation period. TOC was analyzed at day 0 (fresh) and on day 2 (spent) sample from all the loading rates. Immobilisation in the control group was 0 % and dissolved oxygen concentration at the end of the test was higher than 7.32 mg/L in the control and treatment groups. Based on these results, the 48 hours No Observed Effect Loading rate (NOELR) and Lowest Observed Effect Loading rate (LOELR) is 4.8 and 8.6 mg/L respectively, and the acute median effective loading rate (EL50) is 14.95 mg/L (95% confidence limits: 13.34 - 16.77 mg/L).

Description of key information

Key study. Test method according to OECD 202, GLP study. The 48h-EL50 of the test substance to Daphnia magna was found to be 14.95 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

14.95 mg/L