Bis(isopropyl)naphthalene

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

27 Mar 2020 - 03 Jul 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Analytical samples were analysed from all test item concentrations, control and solvent control from days 0, 7, 12, 14 and 21 (one representative vessel of each treatment group). Analytical samples were analysed from day 1 from test item concentrations of 10.0 and 40.0 µg/L (one representative vessel of each treatment group) and from day 12 from all four parallel replicates of the highest test item concentration to prove homogenous dosing. Analytical samples taken from the stock solutions, when freshly prepared and from aged stock solutions. The maximum storage period from sampling to analysis was 8 days within this study. For the storage stability recovery extracts were reanalysed after 38 d of storage at < - 18 °C. The determined mean value was 103% of the 0 d at storage.

Vehicle:

yes

Remarks:

Acetone

Details on test solutions:

The stock solutions for flow-through were mixed thoroughly by shaking until the test item was dissolved in acetone.
For each concentration 65 µL/h of the appropriate stock solution was dosed by the syringe pump into mixing vessels (250 mL bottles placed on a stirrer). In parallel 10.8 mL/min of test medium was dosed into the mixing vessels using piston pumps.
From the mixing vessels the solutions were divided into each replicate with teflon tubes using peristaltic pumps. The dosing per replicate was approximately 2.71 mL/ min.
The concentration of the solvent acetone was 100 µL/L test solution in all test concentrations and in the solvent control.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Strain/clone: Clone V
- Age at study initiation: Less than 24 hours
- Source: Federal Environment Agency in Berlin, Germany
- Feeding during test : Yes
- Frequency: Continuously
- Food type: Algae (Desmodesmus subspicatus) suspension & nutrients (Tetra Min Baby®)
- Amount: according to the age of the daphnids: 0.1 mg carbon / Daphnia / day from day 0 to 7; 0.15 mg carbon / Daphnia / day from day 8 to 14; 0.2 mg carbon / Daphnia / day from day 15 to 21.
Additional nutrients (Tetra Min Baby®) were fed continuously from day 0 to 21. The feed was mixed with test medium and continuously provided. Daphnia were additionally fed with a suspension of algae directly into each test vessel at test start and from day 11 onwards, as it was recognized that feeding was not sufficient due to the daily water exchange (no formation of algae ground covering possible).

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

13 °dH

Test temperature:

20.8 – 21.6 °C

pH:

7.51 – 7.92

Dissolved oxygen:

≥ 6.5 mg/L (7.8 mg/L mean)

Nominal and measured concentrations:

nominal: 40.0, 20.0, 10.0, 5.00 and 2.50 µg/L, solvent control and control
measured (TWA): 24.4, 13.2, 7.2, 3.0, 1.70 µg/L, solvent control and control

Details on test conditions:

TEST SYSTEM
- Test vessel: SCHOTT flasks
- Type: closed with screw cap HPLC, GL 45, 4 ports, complete with silicone seals
- Material, headspace, fill volume: Glas, 500 mL, no headspace
- Aeration: none
- Type of flow-through: peristaltic pumps (flow rates of dilution water) / piston pumps (flow rates stock solutions)
- Renewal rate of test solution (frequency/flow rate): 2.71 mL/min per replicate corresponding to 3.9 L/24 h was used (exchange of ~ 7.8 test chamber volumes per 24 h).
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Elendt M4 medium
- Intervals of water quality measurement: Temperature, pH, oxygen cocnentration and total hardness were measured after 0,7,14 and 21 days.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours photoperiod /8 hours darkness daily
- Light intensity: 1058 - 1123 lux

EFFECT PARAMETERS MEASURED:
- Mortality, reproductivity, body length

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: No range-finding test was performed

Reference substance (positive control):

no

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 40 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mortality, reproduction and body length

Remarks on result:

other: highest test concentration

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 24.4 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: mortality, reproduction and body length

Remarks on result:

other: highest test concentration

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 40 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mortality, reproduction and body length

Remarks on result:

other: highest test concentration

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 24.4 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: mortality, reproduction and body length

Remarks on result:

other: highest test concentration

Details on results:

Mortality of adult Daphnia was observed, but did not follow a monotonous concentration-effect curve and thus is not considered to be concentration-related. At the end of the test the mortality at the highest test item concentration of 40.0 µg/L (nominal) equivalent to 24.4 µg/L (actual) was within the range of the allowed control mortality.

Reported statistics and error estimates:

The LOEC is the lowest test item concentration tested showing a statistically significant difference from the control for the respective endpoint. The NOEC is the highest test item concentration tested below the LOEC for the respective endpoint. The LC50/EC50 is the test item concentration causing 50% variation in the respective endpoint of the test organism population. Even as the control and solvent control did not show statistically significant difference for the parameter reproduction, the evaluation of endpoints was performed comparing the test item concentrations to the solvent control.
The NOEC and LOEC for mortality was observed.Replicate I of the test item concentration of 10.0 μg/L was not considered for the evaluation, since all Daphnia in this replicate died on the same day without any traceable reason. For the calculation of NOEC and LOEC of reproduction and body length, SAS® Proprietary Software Version 9.4 was used. No statistical evaluation of EC50/LC50 was performed, since reduction of reproduction, mortality and body length was found to be below 50% for all concentration levels.

Analytical Results:

The measured content of 1,3-Diisopropylnaphthalene was between 42 and 86% of nominal with a mean of 65% of nominal. The measured content of 1,3-Diisopropylnaphthalene in all four parallel replicates of the highest test item concentration on day 12 was between 58 and 62% of nominal and proved homogeneous dosing. The measured content of 1,3-Diisopropylnaphthalene was between 116 and 136% of nominal in the fresh stock solutions and between 117 and 135% of nominal in the aged stock solutions. It is therefore proven that stock solutions remained stable between renewals.

Table 1: Validity criteria for OECD 211.

Criterion from the guideline	Outcome	Validity criterion fulfilled
The mortality of the parent animals in the control (female Daphnia) does not exceed 20% at the end of the test.	5 %	yes
The mean number of living offspring produced per parent animalsurvivingat the end of the test is ≥ 60 in the control.	63	yes

Table 2: Number of adult Daphnia and offspring

Conc./Day	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21	Σ
Control
Adults	39	39	39	39	39	39	39	39	39	39	39	39	39	38	38
Offspring alive	0	176	31	78	50	23	18	141	55	100	294	149	562	350	365	2392
Offspring dead	0	0	0	0	0	0	0	0	0	0	0	0	0	1	0	1
Solvent
Adults	39	39	39	39	39	39	39	39	39	39	38	38	37	37	37
Offspring alive	10	264	69	92	183	50	9	212	64	241	312	55	801	352	129	2843
Offspring dead	0	0	0	0	0	0	0	0	0	1	0	0	0	0	0	1
2.50 µg/L
Adults	38	38	38	38	38	38	38	37	37	36	36	35	35	35	35
Offspring alive	17	220	68	82	176	37	72	101	75	224	164	186	604	330	521	2877
Offspring dead	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
5.00 µg/L
Adults	40	40	40	40	40	40	40	40	40	40	39	39	38	38	38
Offspring alive	13	201	101	30	139	60	9	110	94	149	205	222	464	399	303	2499
Offspring dead	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
10.0 µg/L*
Adults	40	30	30	30	30	30	30	30	30	30	30	30	30	30	30
Offspring alive	25	210	27	17	141	20	4	109	42	92	299	88	581	194	210	2051
Offspring dead	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
20.0 µg/L
Adults	39	39	39	39	39	39	39	39	39	39	39	39	39	39	38
Offspring alive	44	350	35	32	232	16	77	128	27	501	110	43	1106	94	331	3126
Offspring dead	0	0	0	0	0	0	1	0	0	0	0	0	0	0	0	1
40.0 µg/L
Adults	38	38	37	37	37	37	37	37	37	37	37	37	37	37	37
Offspring alive	107	282	15	98	170	0	58	84	8	396	72	32	922	53	427	2724
Offspring dead	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0

Table 3: Mortality of adult animals (% of inital numbers)

Day / Conc. [µg/L]	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21
Control	0	0	0	0	0	0	2.5	2.5	2.5	2.5	2.5	2.5	2.5	2.5	2.5	2.5	2.5	2.5	2.5	5.0	5.0
Solvent Control	0	0	2.5	2.5	2.5	2.5	2.5	2.5	2.5	2.5	2.5	2.5	2.5	2.5	2.5	2.5	5.0	5.0	7.5	7.5	7.5
2.50	0	0	0	0	5.0	5.0	5.0	5.0	5.0	5.0	5.0	5.0	5.0	7.5	7.5	10.0	10.0	12.5	12.5	12.5	12.5
5.00	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	2.5	2.5	5.0	5.0	5.0
10.0*	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
20.0	0	0	0	0	2.5	2.5	2.5	2.5	2.5	2.5	2.5	2.5	2.5	2.5	2.5	2.5	2.5	2.5	2.5	2.5	5.0
40.0	0	0	5.0	5.0	5.0	5.0	5.0	5.0	7.5	7.5	7.5	7.5	7.5	7.5	7.5	7.5	7.5	7.5	7.5	7.5	7.5

 

Table 4: Determined concentration of 1,3 -DIPN

Test item nominal (μg/L)	1,3-Diisopropylnaphthalene nominal (μg a.i./L)	Sampling	1,3-Diisopropylnaphthalene found		Time weighted arithmetic mean [%]	Test item actual [μg/L] 1)
			μg a.i./L	% of nominal
Control	0	0 d	< LOQ	-	-	-
		7 d	n.d.	-
		12 d	n.d.	-
		14 d	n.d.	-
		21 d	n.d.	-
Solvent Control	0	0 d	n.d.	-	-	-
		7 d	n.d.	-
		12 d	n.d.	-
		14 d	n.d.	-
		21 d	n.d.	-
2.50	2.50	0 d	1.87	75	68	1.70
		7 d	1.48	59
		12 d	1.52	61
		14 d	1.89	76
		21 d	1.81	72
5.00	5.00	0 d	4.31	86	60	3.00
		7 d	2.09	42
		12 d	2.88	58
		14 d	3.19	64
		21 d	3.10	62
10.0	10.0	0 d	5.78	58	72	7.20
		1 d	5.82	58
		7 d	7.54	75
		12 d	6.84	68
		14 d	7.93	79
		21 d	7.95	80
20.0	20.0	0 d	14.5	73	66	13.2
		7 d	12.9	65
		12 d	11.7	59
		14 d	12.2	61
		21 d	14.5	73
40.	40.0	0 d	18.2	46	61*	24.4
		1 d	26.4	66
		7 d	22.5	56
		12 d (Replicate 1)	24.2	61
		12 d (Replicate 2)	23.7	59
		12 d (Replicate 3)	23.1	58
		12 d (Replicate 4)	24.7	62
		14 d	23.9	60
		21 d	27.1	68

- = not calculated; n.d. = not detectable; LOQ = 0.250 μg a.i./L; LOD = 0.0750 μg a.i./L

1) Actual of test item based on time-weighted arithmetic mean of 1,3-Diisopropylnaphthalene

*on day 12 the mean of the 4 replicated was taken to calculate the time-weighted arithmetic mean

Validity criteria fulfilled:

yes

Remarks:

For more details see table 1 under "any other information on results inc. tables"

Reference 2

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

08 Jun – 08 Jul 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

October 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Analytical samples were analysed from all test item concentrations, control and solvent control from days 0, 7, 14 and 21 (one representative vessel of each treatment group). Analytical samples were analysed from day 1 from test item concentrations of 7.81, 15.6, 31.3, 62.5 and 125 µg/L (one representative vessel of each treatment group) and from day 14 from all four parallel replicates of the highest test item concentration to prove homogenous dosing. Analytical samples taken from the stock solutions, when freshly prepared and from aged stock solutions.
Storage stability: After 30 days of storage (deep frozen (≤ 18 °C); the mean of recovery sample extracts were 101% of the day 0 value. This time period covers the longest storage period of sample extracts of 8 days.

Vehicle:

yes

Remarks:

Acetone

Details on test solutions:

The stock solutions for flow-through were mixed thoroughly by shaking until the test item was dissolved in acetone.
For each concentration 65 µL/h of the appropriate stock solution was dosed by the syringe pump into mixing vessels (250 mL bottles placed on a stirrer). In parallel 10.8 mL/min of test medium was dosed into the mixing vessels using piston pumps.
From the mixing vessels the solutions were divided into each replicate with teflon tubes using peristaltic pumps. The dosing per replicate was approximately 2.71 mL/ min.
The concentration of the solvent acetone was 100 µL/L test solution in all test concentrations and in the solvent control.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Strain/clone: Clone V
- Age at study initiation: Less than 24 hours
- Source: Federal Environment Agency in Berlin, Germany
- Feeding during test: Yes
- Frequency: Continuously
- Food type: Algae (Desmodesmus subspicatus) suspension & nutrients (Tetra Min Baby®)
- Amount: According to the age of the daphnids: 0.1 mg carbon / Daphnia / day from day 0 to 7; 0.15 mg carbon / Daphnia / day from day 8 to 14; 0.2 mg carbon / Daphnia / day from day 15 to 20;
Additional nutrients (Tetra Min Baby®) were fed continuously from day -2 to 20; the feed was mixed with test medium and continuously provided.
Daphnia were additionally fed with a suspension of algae and Tetra Min Baby® directly into each test vessel each day until day 20 of the test, as due to the daily water exchange no formation of algae ground covering was possible.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

13 °dH (control, solvent control and all test item concentrations)

Test temperature:

20.8 – 21.5 °C

pH:

7.42 – 7.94

Dissolved oxygen:

8.5 - 9.3 (Control)
7.7 - 8.0 (Solvent control)
7.7 - 8.7 (Test item concentrations)

Nominal and measured concentrations:

Control, Solvent control, 7.81, 15.6, 31.3, 62.5 and 125 µg/L (nominal)
0,0, 3.83, 8.11, 16.3, 28.1 and 38.8 µg/L (measured arithmetic mean)

Details on test conditions:

TEST SYSTEM
- Test vessel: SCHOTT flasks
- Type: closed with screw cap HPLC, GL 45, 4 ports, complete with silicone seals
- Material, headspace, fill volume: Glas, 500 mL, no headspace
- Aeration: none
- Type of flow-through: peristaltic pumps (flow rates of dilution water) / piston pumps (flow rates stock solutions)
- Renewal rate of test solution (frequency/flow rate): 2.71 mL/min per replicate corresponding to 3.9 L/24 h was used (exchange of ~ 7.8 test chamber volumes per 24 h).
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Elendt M4 medium
- Intervals of water quality measurement: Temperature, pH, hardness and oxygen concentration were measured at day 0,7, 14 and 21

OTHER TEST CONDITIONS
- Photoperiod: 16 hours photoperiod /8 hours darkness daily
- Light intensity: 1099 - 1219 lux

EFFECT PARAMETERS MEASURED:
- Mortality, reproductivity, body length

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
Range finding study: No range-finding test was performed

Reference substance (positive control):

no

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

62.5 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: based on total number of living offspring produced per parent present at the beginning of the test

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

28.1 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: based on total number of living offspring produced per parent present at the beginning of the test

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

125 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: based on total number of living offspring produced per parent present at the beginning of the test

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

38.8 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: based on total number of living offspring produced per parent present at the beginning of the test

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

31.3 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

16.3 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

62.5 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

28.1 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

In the solvent control, the control and at test item concentrations of 7.81, 15.6 and 31.3 µg/L no mortality above the allowed control mortality was observed. At the test item concentration of 62.5 µg/L 22.5 % mortality was observed. At the highest test item concentration of 125 µg/L 92.5 % mortality was observed. Mortality of adult Daphnia was observed and did follow a monotonous concentration-effect curve; thus mortality is considered to be concentration-related.
The body length of adults alive at test end did not show any statistically significant inhibition in any test item concentration compared to the solvent control. The LOEC was supposed to be >125 µg/L (nominal) equivalent to >38.8 µg/L (actual). The NOEC was set to be ≥125 µg/L (nominal) equivalent to ≥38.8 µg/L (actual). The EC50 could not be calculated due to effects below 10% and is estimated to be above the maximum test concentration.

Reported statistics and error estimates:

The NOEC and LOEC for reproduction were determined by using a multiple comparison method (Dunnetts-t-Test, left-sided, p < 0.05) of the SAS® Proprietary Software Version 9.4.. The significance level for all statistical tests was p < 0.05.

Table 1: Validity criteria

Criterion from the guideline	Outcome	Validity criterion fulfilled
The mortality of the parent animals in the control (female Daphnia) does not exceed 20% at the end of the test.	0% (Control) 15% (Solvent Control)	Yes
The mean number of living offspring produced per parent animalsurvivingat the end of the test is ≥ 60 in the control.	147 (Control) 163 (Solvent Control)	Yes

Table 2: Mortality of adult animals for every day (% of initial numbers) 

Day / Conc. [µg/L]	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21
Control	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
SolventControl	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	5.0	5.0	5.0	5.0	5.0	7.5	7.5	12.5	15.0
7.81	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	5.0	7.5	7.5	7.5	7.5	7.5	7.5	7.5
15.6	0.0	0.0	0.0	0.0	0.0	2.5	2.5	2.5	2.5	2.5	2.5	2.5	5.0	5.0	5.0	5.0	5.0	5.0	5.0	5.0	15.0
31.3	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
62.5	0.0	0.0	0.0	0.0	0.0	0.0	0.0	2.5	2.5	2.5	2.5	5.0	5.0	5.0	5.0	5.0	7.5	10.0	12.5	15.0	22.5
125	0.0	0.0	0.0	0.0	5.0	7.5	10.0	42.5	65.0	65.0	65.0	67.5	67.5	67.5	70.0	82.5	85.0	90.0	92.5	92.5	92.5

Table 3: Number of adult Daphnia and offspring

Conc./Day	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21	Σ
Control
Adults	40	40	40	40	40	40	40	40	40	40	40	40	40	40	40
Offspring alive	797	37	1	1464	3	175	1370	3	57	869	20	52	936	33	81	5898
Offspring dead	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Solvent
Adults	40	40	40	40	40	40	38	38	38	38	38	37	37	35	34
Offspring alive	860	61	102	1198	66	490	962	67	197	633	59	207	609	63	172	5746
Offspring dead	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
7.81 µg/L
Adults	40	40	40	40	40	40	40	38	37	37	37	37	37	37	37
Offspring alive	696	96	84	1277	51	337	1167	35	149	750	27	128	680	50	172	5699
Offspring dead	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
15.6 µg/L
Adults	39	39	39	39	39	39	38	38	38	38	38	38	38	38	34
Offspring alive	829	59	195	976	3	845	533	2	565	381	3	377	362	7	312	5449
Offspring dead	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
31.3 µg/L
Adults	40	40	40	40	40	40	35	35	34	34	34	33	33	33	33
Offspring alive	773	55	94	1167	30	377	1078	43	239	605	27	170	581	25	186	5450
Offspring dead	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
62.5 µg/L
Adults	40	39	39	39	39	38	38	38	38	38	37	36	35	34	31
Offspring alive	783	21	40	1190	0	732	637	2	589	344	0	649	158	2	619	5766
Offspring dead	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
125 µg/L
Adults	36	23	14	14	14	13	13	13	12	7	6	4	3	3	3
Offspring alive	275	39	34	198	2	144	118	0	36	30	0	64	26	0	103	1069
Offspring dead	0	4	0	0	0	0	0	0	0	0	0	0	0	0	0	4

 

Table 4: Determined concentration of 1,4-Diisopropylnaphthalene

Test item nominal [µg/L]	1,4-Diisopropylnaphthalene nominal [µg a.i./L]	Sampling	1,4-Diisopropylnaphthalene found		Time-weighted arithmetic mean [%]	Test item actual   [µg/L] 1)
			[µg a.i./L]	% of nominal
Control	0	0 d	n.d.	-	-	-
		7 d	n.d.	-
		14 d	n.d.	-
		21 d	n.d.	-
Solvent Control	0	0 d	n.d.	-	-	-
		7 d	n.d.	-
		14 d	n.d.	-
		21 d	n.d.	-
7.81	7.81	0 d	3.79	49	49	3.83
		7 d	3.58	46
		14 d	4.27	55
		21 d	3.23	41
15.6	15.6	0 d	7.51	48	52	8.11
		7 d	8.92	57
		14 d	7.91	51
		21 d	7.43	48
31.3	31.3	0 d	20.9	67	52	16.3
		7 d	15.1	48
		14 d	16.8	54
		21 d	12.9	41
			35.0	56
62.5	62.5	0 d	35.0	56	45	28.1
		7 d	30.4	49
		14 d	23.4	37
		21 d	25.6	41
125	125	0 d	60.9	49	31*	38.8
		7 d	44.2	35
		14 d (Replicate 1)	30.4	24
		14 d (Replicate 2)	27.7	22
		14 d (Replicate 3)	30.5	24
		14 d (Replicate 4)	33.8	27
		21 d	19.5	16

LOQ = 0.781 µg a.i./L; LOD = 0.234 µg a.i./L

- = not calculated; n.d. = not detectable; LOQ = 0.781 µg a.i./L; LOD = 0.234 µg a.i./L

1) Actual of test item based on time-weighted arithmetic mean of 1,4-Diisopropylnaphthalene

*on day 14 the mean of the 4 replicated was taken to calculate the time-weighted arithmetic mean

Validity criteria fulfilled:

yes

Remarks:

For more details see table 1 under "any other information on results inc. tables"

Reference 3

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

yes

Remarks:

The temperature was 19.2 to 23.2°C instead of 18.0 - 22.0°C, the difference was more than 2 °C.

Qualifier:

according to guideline

Guideline:

EU Method C.20 (Daphnia magna Reproduction Test)

Deviations:

yes

Remarks:

The temperature was 19.2 to 23.2°C instead of 18.0 - 22.0°C, the difference was more than 2 °C.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

TEST MATERIAL:
- Composition: diisopropyl naphthalene, mixture of isomers (see report: attached certificate of analysis)
- Appearance: colourless liquid

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 9124008508 (by RÜTGERS Novares GmbH, Duisburg, Germany)
- Production date: 17. July 2015
- Expiration date of the lot/batch: 31. Dec. 2017
- Purity test date: 99.4% (gas chromatography)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: closed vessel, room temperature (20 ± 5°C), darkness
- Stability under test conditions:
- Solubility of the test substance in solvent: H2O: 0.12 - 0.15 mg/L; EtOH: > 1 g/L; acetone: > 1 g/L; CH3CN: > 1 g/L; DMSO: > 1 g/L
- Stability of the test substance in solvent: H2O: volatile; EtOH: 96h; acetone: 96h; CH3CN: 96h; DMSO: 96h

FORM AS APPLIED IN THE TEST : Applied as water accomodated fraction (WAF)

OTHER SPECIFICS:
Density: 0.958 g/kg
Homogeneity: homogeneous (>-15°C)
Volatility: 0.005 hPa at 20°C

Analytical monitoring:

yes

Details on sampling:

The content of the test item in the solutions was measured several times in the freshly prepared and in the used test solutions after 48 hours and 72 hours.
Additionally, at 39 µg/L and 16 µg/L, the concentrations were analysed in the old solutions in the absence of food (algae) and test organism (daphnia) in order to compare with the corresponding terminal test item levels in the presence of food and daphnia.

Sampling Date# Day 0 Day 2 Day 4 Day 7 Day 9 Day 11 Day 14 Day 16 Day 18 Day 21
Renewal 1 Renewal 2 Renewal 3 Renewal 4 Renewal 5 Renewal 6 Renewal 7 Renewal 8
Start 48h 48h 72h 48h 48h 72h 48h 48h End
==================================================================================================================================================
Treatment Mo We Fr Mo We Fr Mo We Fr Mo
----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Blank control new old new old new old -- -- new old
60 µg/L new old new old new old -- -- new old
39 µg/L new old (2) new old (2) new old (2) -- -- new old (2)
25 µg/L new old new old new old -- -- new old
16 µg/L new old (2) new old (2) new old (2) -- -- new old (2)
10 µg/L new old new old new old -- -- new old
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
*39 µg/L -- old -- old -- old -- -- old
*16 µg/L -- old -- old -- old -- -- old
===================================================================================================================================================
* additional replicates without daphnia and algae, only for analytical measurement
# In the last row of the headline, the elapsed time since the last renewal is mentioned.
New = fresh medium, old = used medium (after 48 or 72 h), (2) = analysis of two replicates per treatment

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
A saturated solution (WAF) using the loading rate of 10 mg/L test item was prepared for the test.
8.4 µL of test item was added to 800 mL of M4 medium (on the surface of the medium, while stirring on a magnetic stirrer).
The solutions were slowly stirred at room temperature under sealed conditions for 24 h. After this time, 400 mL of the lower water phase was separated after discarding of approx. 20 mL forerun.
At the start of the test and for each medium renewal, 5 flasks with saturated solution were prepared. The resulting solutions were mixed to one batch.
The lower test concentrations (treatments) were prepared by diluting the saturated solution with dilution water.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna, female (multipied by parthenogenesis)
- Strain/clone: STRAUS
- Source: LAUS GmbH (originally obtained from the Umweltbundesamt, Berlin)
- Age at test start: < 24 hours
- Vessels: Glass beakers, nominal volume 2 litres
- Medium: M4-Medium (recipe of ELENDT), composition see report, annex 5
- Food: Green alga (Desmodesmus subspicatus)
- Medium renewal: twice a week
- Photo-period: 16/8 hours, using neon tubes
- Temperature: 20 ± 2 °C
- Feeding during test: Suspension of unicellular green algae Desmodesmus subspicatus, 0.1 - 0.2 mg organic carbon (OC)/animal/day, adjusted to 3 feeding points per week

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
24 hours before the start of the test, the adult animals were separated from the young. 23 hours later, the adults were caught with the help of a glass tube, and the newborn daphnia, aged between 0 and 23 hours, were sieved from the medium and immediately placed into a 250 mL-beaker containing dilution water. After a settling-in period of 30 minutes, animals which showed no apparent damage were used for the test.
Using a glass tube, the daphnia were caught and lifted from the beaker. They were put on a small sieve, and the medium surrounding the animals was sucked off using absorbent paper. Immediately after that, the animals were put into the respective test solution.
Then the test vessels were closed immediately. To avoid loss of test item, the test vessels were inspected for offspring only three times a week at each medium renewal. Then, the newborn daphnia were counted and removed from the test vessel.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

267 - 356 mg CaCO3/L (for details see table below)

Test temperature:

19.2°C - 23.2°C

pH:

7.2 - 8.9 (for details see table below)

Dissolved oxygen:

8.9 - 11.6 mg/L (for details see table below)

Salinity:

no data

Conductivity:

no data

Nominal and measured concentrations:

five target concentrations: 10, 16, 25, 39, 60 µg/L (nominal)
Analytically determined concentrations: see table below

Details on test conditions:

TEST SYSTEM
- Test vessel: Closed 100 mL SCHOTT flasks with PTFE seals
- Type: closed
- Aeration: none
- Renewal rate of test solution: three times a week, using freshly prepared test solutions
- At each medium renewal, the newborn daphnia were counted and removed from the test vessel.
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- Blank control: Dilution water (10 replicates)
- Additional batch without algae and daphnia for analytical measurement of the old solutions only: Treatment 16 µg/L and 39 µg/L (nominal).

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Deionised water enriched with minerals. The minerals which are added comply with the composition M4 (full composition see report, annex 5,) derived by ELENDT as stated in the OECD-Guideline
- pH, oxygen and hardness were measured in the new and the old test solutions (blank control and highest test concentration) once per week

OTHER TEST CONDITIONS
- Adjustment of pH: no data
- Photoperiod: 24 h
- Light intensity: 1000 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Mortality, reproduction (Number of surviving offspring)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: The concentrations to be tested are based on the results of a non-GLP pre-test without analytical determination
- Test concentrations: In the two highest concentrations 100 % mortality was observed, whereas in the two lower concentrated treatments the offspring of the surviving animals was in the same range as in the control.
Therefore, the main test was performed using five target concentrations ranging from 60 to 10 µg/L.

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

11.8 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

12 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

14 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

17 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

33 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

- For detailed results for reproduction/ fecundity see tables below and report, annex 2
- Dose response: see figures (attached document)
- Mortality: The two highest test concentrations (60 and 39 µg/L) showed 100 % mortality.
- Test solutions: In spite of the low test concentrations and the volatile test item, the measured concentrations were in the expected range and showed good reproducibility.
In the old solutions without daphnia and algae, the measured concentrations were distinctly higher than in the solution with test organism. The measured concentrations in the solutions without test organism were not used for evaluation.

Reported statistics and error estimates:

Calculation of results was performed with the help of validated software (Microsoft Excel®).
The estimation of the biological results was accomplished using the software ToxRat® Professional, version 3.2.1.
Biological results were stated using two significant digits.
The details on calculation are stated in the report, annex 6

Reproduction

Table 8.1-a (excerpt from report): Cumulated number of offspring within 21 days of exposure for each of the 10 replicates per test concentration

Replicate	BlankControl	Target and analytical concentrations [µg/L]
		10	16	25	39	60
		7.6	12	17	29	42
1	75	78	91	94	M	M
2	74	M	90	M	M	M
3	79	98	65	80	M	M
4	61	100	71	90	M	M
5	71	M	59	75	M	M
6	106	90	77	53	M	M
7	104	71	72	48	M	M
8	113	76	93	M	M	M
9	77	78	M	50	M	M
10	77	88	92	60	M	M
Mean	84	85	79	69	--	--
SD	17	11	13	18	--	--
RSD	21	13	16	27	--	--

Mean = per surviving parent animal over 21 days

SD = standard deviation;

RSD = relative standard deviation = coefficient of variation;

M = mortality of parent animal

Table 8.2-a (excerpt from report): Fecundity

Target concentration [µg/L]	Total Offspring of Surviving Parent Animals	Surviving Parent Animals	Mean Offspring per Surviving Parent Animal	Inhibition of Reproduction [%]
Blank Control	837	10	84	0
10	679	8	85	-1
16	710	9	79	6
25	550	8	69	18
39	0	0	--	--
60	0	0	--	--

Validity criteria fulfilled:

yes

Reference 4

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

Investigation of the effect of diisopropylnaphthalene on the reproductive output of the marine copepod Acartia tonsa during 96 h under semi-static conditions (renewal period 24 h). The method is based on the PARCOM protocol (UK proposal to ISO, TC147/SC5/WG2, Nov. 1990) and it is an adaption of the work of Girling (1989, Bull Environ Contam Toxicol 43, 280-286) and Toudal and Riisgard (1987, Mar Ecology 37, 141-146).

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
Samples were taken from saturated solution in marine solution water (100%), from controls (0%) and from 10%, 18%, 32%, and 56% dilutions of saturated solution at t=0h (new medium) and t=24h (used medium).
- Sampling method: not reported, ca. 100 mL test solution per test concentration were sampled for analysis.
- Sample storage conditions before analysis: no storage, samples directly used for analysis

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Method: generator column technique
A saturated solution of test substance in water was prepared by a generator column technique at room temperature. Test substance was coated on column packing material (Chrompack - Chromosorb 60/80 mesh) and a stainless steel HPLC column (ca. 25cm x 5 mm) was filled with the coated material. Natural sea water as dilution water was purged through the column for an extended period of time resulting in a saturated solution. Additional test solutions were prepared by diluting the saturated solution.
- Controls: controls were prepared by treating natural seawater the same way but without using test substance.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): all test solutions were clear by sight.

Test organisms (species):

other: Acartia tonsa (marine)

Details on test organisms:

TEST ORGANISM
- Common name:
- Strain/clone: no data
- Justification for species other than prescribed by test guideline: no data
- Source: Water Quality Institute, Horsholm, Denmark
- Age of parental stock: female animals selected from a population between 12 and 13 days old were used
- Feeding during test
- Food type: Cryptomonas baltica
- Amount: 1.5 x 10E4 cells per mL of test medium
- Frequency: daily

Test type:

semi-static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

19.0 - 19.4°C

pH:

8.1

Dissolved oxygen:

≥7.2 mg/L

Salinity:

29‰

Nominal and measured concentrations:

Nominal concentrations: saturated solution (100%) in natural sea water and 10%, 18%, 32% and 56% dilutions
Measured concentrations: at t=0h (new medium) saturated solution 78 µg/L, 18% dilution 11 µg/L, 32% dilution 20 µg/L, 56% dilution 39 µg/L
at t=24 h (used medium prior to renewal) saturated solution 9.1 µg/L, 18% dilution < 3 µg/L, 32% dilution 3.5 µg/L, 56% dilution 3.7 µg/L
In all initial test media, the concentration of DIPN was analysed to be <3 µg/L after 48 h (before renewal of test media).

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: scintillation vials covered with a perspex plate, size ca. 20 mL, fill volume 17 mL
- Aeration: no
- Renewal rate of test solution (frequency/flow rate): 24 h, semi-static system
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10; blank control (test medium without test substance
- No. of vessels per vehicle control (replicates): 10; column blank (test medium prepared by column treatment of sea water without coating the column fill material with test substance)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: natural sea water originating from the Eastern Scheldt filtered through a sand filter and prior to use over a carbon filter and a 3 and 0.45 µm membrane filter. Salinity was adjusted from 33 to 29‰ with distilled water.
- Culture medium different from test medium: no
- Intervals of water quality measurement: upon arrival

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light / 8 h dark (two transition periods of 0.5 h of twilight)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.02 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: reproduction and mortality

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

0.039 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: reproduction and mortality

There were no adverse effects observed with regard to reproduction, survival or condition of the test animals up to 32 % saturated solution = ~20 µg/l (NOEC). Above, there was a concentration and time-related decrease in survival.

 

 

Survival of test animals and exposure time related LC50 values

 

Time [h]	Number of surviving animals					LC50 at time t
	Exposure level
	control	11 µg/L (18% dilution)	20 µg/L (32% dilution)	39 µg/L (56% dilution)	78 µg/L (100%)
0	10	10	10	10	10
24	10	10	10	10	6	> 78 µg/L
48	10	10	10	8	1	ca. 53 µg/L
72	10	10	10	6	1	ca. 44 µg/L
96	10	10	10	4	0	ca. 37 µg/L

 

Validity criteria fulfilled:

yes

Remarks:

for the range of NOEL

Conclusions:

Diisopropylnaphthalene is toxic to the marine copepod crustacean Acartia tonsa at concentrations of >20 µg/L. Above, a concentration and time related decrease in survival can be observed, thus preventing the independent observation of the effect on reproduction. A NOEC of 20 µg/L for reproduction and mortality was determined in this study.

Reference 5

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Qualifier:

according to guideline

Guideline:

other: OECD test guideline 202, part 2

Deviations:

yes

Remarks:

: Reproduction rate approx. 10 % less than required 60 offspring per female / Chemical analysis only in stock solution

GLP compliance:

no

Analytical monitoring:

yes

Remarks:

Only stock solution was analysed for test substance concentration.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Preparation of a water-soluble fraction: 0.5 mg/L test substance in dilution water was stirred for 48 h and then filtrated over glass fiber filters.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Strain: IRCHA
- Source: no data
- Age of parental stock (mean and range, SD): < 24 h
- Feeding during test
- Food type: Tetramin (breeding feed for young fish) and and activated sludge (autoclaved)
- Amount: 15 - 20 mg/L CSB per vessel (5 animals)
- Frequency: daily

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Test temperature:

20 ± 1°C (reported value of 25 is very likely a misprint)

pH:

7.1 - 7.4

Dissolved oxygen:

68 - 83% saturation

Nominal and measured concentrations:

Test solutions
nominal concentrations: 0.002, 0.003, 0.006, 0.013, 0.025, 0.050, 0.1, and 0.2 mg/L
Stock solution
nominal concentration: 0.5 mg/L
measured concentration:mean 0.29 mg/L, S.D. 0.085, range 0.19 - 0.44 (analysis of stock solution on 9 occasions during test period)

Details on test conditions:

TEST SYSTEM
- Test vessel: no data
- Fill volume per test vessel: 250 mL
- Renewal rate of test solution (frequency/flow rate): three times a week
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 8

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: synthetic medium according to DIN 38412, part 11
- Intervals of water quality measurement: three times per week

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 9 h light / 15 h dark
- Light intensity: ca. 300 LUX

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): live offspring, mortality (three times a week)

Reference substance (positive control):

no

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.013 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: reproduction rate and mortality

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

0.025 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: reproduction rate and mortality

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

ca. 0.025 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

- Mortality of parent animals: yes, see table under Remarks on results: Concentration-related increase in mortality with concomitant decrease in offspring

Reported statistics and error estimates:

The U-test and t-test were applied to describe differences in the reproduction rate and mortality.

Test Results Summary Table

 

Concentration (nomimal)	Reproduction rate		Mortality
[mg/L]	Av. RR	VK	D/T	Av. M [%]	VK
0 (control)	54.9	2.2	0/40	0	0.0
0.002	59.1	3.4	0/20	0	0.0
0.003	52.9	3.7	2/20	10	57.7
0.006	55.1	3.3	2/20	10	57.7
0.013	50.9	7.3	0/20	0	0.0
0.025	41.6*	4.1	12/20	60*	30.4
0.050	27.0*	7.2	18/20	90*	11.1
0.100	0.0*	0.0	20/20^1)	100*	0.0
0.200	0.0*	0.0	20/20^1)	100*	0.0

 

Av. RR = average reproduction rate per female

D/T      = Dead animals out of total animals

Av. M  = mortality in %

VK        = variance coefficient

 

* p ≤ 0.05 (U-test)

¹⁾ at day 5

Validity criteria fulfilled:

no

Remarks:

Reproduction rate per adult daphnia approx. 10 % below required 60 neonates per animal.

Reference 6

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Acceptable, well-documented study which meets basic scientific principles, but not fully evaluable due to poor stability of the substance concentrations

Qualifier:

no guideline followed

Principles of method if other than guideline:

Investigation of the effect of diisopropylnaphthalene on the reproductive output of the marine copepod Acartia tonsa during 96 h under semi-static conditions (renewal period 48 h). The method is based on the PARCOM protocol (UK proposal to ISO, TC147/SC5/WG2, Nov. 1990) and it is an adaption of the work of Girling (1989, Bull Environ Contam Toxicol 43, 280-286) and Toudal and Riisgard (1987, Mar Ecology 37, 141-146)

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
Samples were taken from saturated solution in marine solution water (100%), controls (0%) and 56%, 18% dilutions of saturated solution and from water soluble fraction at t=0 (new medium) and t=48h (used medium). For 100% saturated solution and for water soluble fraction, samples were also taken from new renewal medium (t=48 h).
- Sampling method: not reported, ca. 100 mL test solution per test concentration were sampled for analysis.
- Sample storage conditions before analysis: no storage, samples directly used for analysis

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Method 1- saturated solution
A saturated solution of test substance in water was prepared by a generator column technique at room temperature. Test substance was coated on column packing material (Chrompack - Chromosorb 60/80 mesh) and an stainless steel HPLC column (ca. 25cm x 5 mm) was filled with the coated material. Natural sea water as dilution water was purged through the column for an extended period of time resulting in a saturated solution. Additional test solutions were prepared by diluting the saturated solution.
Method 2 - water soluble fraction (WSF)
The test medium was prepared by mixing 0.10 g of the test substance with 1 L of natural sea water in the dark for 20 h under mechanical stirring. Phase separation was allowed leaving the mixture stand still for 24 h, before the aqueous phase (WSF) was drawn off.
- Controls: controls were prepared by treating natural seawater the same way but without using test substance.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): all test solutions were clear by sight.

Test organisms (species):

other: Acartia tonsa (marine)

Details on test organisms:

TEST ORGANISM
- Common name:
- Strain/clone: no data
- Justification for species other than prescribed by test guideline: no data
- Source: Water Quality Institute, Horsholm, Denmark
- Age of parental stock (mean and range, SD): female animals selected from a population between 12 and 13 days old were used
- Feeding during test
- Food type: Cryptomonas baltica
- Amount: 1.5 x 10E4 cells per mL of test medium
- Frequency: daily

Test type:

semi-static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

20.2 - 20.5°C

pH:

8.0 - 8.1

Dissolved oxygen:

≥ 7.0 mg/L

Salinity:

29.4‰

Nominal and measured concentrations:

Nominal concentrations: saturated solution (100%) in natural sea water and 10%, 18%, 32% and 56% dilutions; water soluble fraction in sea water
Measured concentrations: saturated solution 61 and 44 µg/L (initial and new 48 h renewal test medium), 18% dilution 13 µg/L, 56% dilution 38 µg/L, water soluble fraction 122 µg/L (initial test medium)
In all initial test media, the concentration of DIPN was analyzed to be <3 µg/L after 48 h (before renewal of test media).

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: scintillation vials covered with a perspex plate, size ca. 20 mL, fill volume 17 mL
- Aeration: no
- Renewal rate of test solution (frequency/flow rate): 48 h, semi-static system
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10 (saturated sea water and water soluble fraction)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: natural sea water originating from the Eastern Scheldt filtered through a sand filter
and prior to use over a carbon filter and a 5 µm membrane filter. Salinity was adjusted from 33 to 29‰ with distilled water.
- Culture medium different from test medium: no
- Intervals of water quality measurement: upon arrival

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light / 8 h dark (two transition periods of 0.5 h of twilight)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): number of eggs and nauplii at 48 h (media refreshment)
and at 96 h (end of test); survival and conditions visually (daily)

Duration:

4 d

Dose descriptor:

NOEC

Effect conc.:

ca. 52 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: highest concentration tested, saturated solution

Details on results:

- Mortality of parent animals:
controls: no mortality
saturated solution - 10, 32, 56% dilutions: no mortality
saturated solution - 100%: 2 parent animals of a total of 10 animals died (between 48 and 72 h)
water soluble fraction: 10 parent animals of a total of 10 animals died (7 and 3 animals still alive after 24 and 48h, respectively)

Reported statistics and error estimates:

Statistical significance of reproduction results was verified using a two tailed Dunnett's test (99% level).

DIPN had disappeared from all test media after 48 h, in the initially saturated solution (61 µg/L) as well as in the water soluble fraction (122 µg/L), which underlines the low residence time of the compound in the aqueous compartment.

20% mortality is considered to be acceptable in a reproduction test (as for Daphnia magna).

 

There were no adverse effects observed with regard to reproduction, survival or condition of the test animals when exposed to the saturated solution or its dilutions.

 

In the water soluble fraction (with higher initial concentration), all of the test animals died within 72 h with 7 and 3 animals still alive after 24 h and 48 h, respectively. The higher initial concentration of the test substance is considered to be most like the reason for this effect.

Validity criteria fulfilled:

yes

Remarks:

according to criteria stated in this report, but see restriction of validity due to instability of DIPN in solution.

Conclusions:

Diisopropylnaphthalene applied as saturated solution in natural sea water (initial concentration 61 µg/L) had no advers effect on reproduction of test animals (copepod Acartia tonsa) under the conditions of this test. A 96 h NOEC of 52 µg/L was determined for the effect on reproduction of the test animals.

Description of key information

In a Daphnia magna reproduction test according to OECD test guideline 211, a NOEC of 0.0118 mg/L (meas. TWA) was determined.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.012 mg/L

Marine water invertebrates

Marine water invertebrates

Effect concentration:

0.02 mg/L

Additional information

Four tests on long-term toxicity of the isomeric mixture bis(isopropyl)naphthalene (CAS 38640-62-9) to aquatic invertebrates are available, two for the freshwater species Daphnia magna (WaBoLu/BGA, 1990 & Laus 2017) and two for the marine copepods Acartia tonsa (TNO, 1998a & 1998b).

One reproduction test with Daphnia magna was conducted according to OECD 211 (Laus, 2017). The Daphnids were exposed to the substance for 21 days in a semi-static test design. The test solutions were renewed three times per week. Based on Daphnia reproduction a NOEC (21 d) of 0.0118 mg/L (measured, TWA) was determined. This result is confirmed by the findings of a second chronic toxicity test on D. magna, conducted according to OECD 202, part 2 (adopted 1984) (WaBoLu/UBA 1990b). The determined NOEC (21 d) in this study was 0.013 mg/L (nominal).

The studies with the marine copepod Acartia tonsa (TNO, 1998a & 1998b) resulted in NOEC values of approx. 0.05 mg/L (measured) and 0.02 mg/L (measured), respectively.

In all four studies a decrease of test item was noted, which might lead to an underestimation of the toxicity.

 

Considering that the results of the two Daphnia reproduction studies with the isomeric mixture bis(isopropyl)naphthalene are on the borderline of meeting the T-criterion, two further Daphnia reproduction tests in a closed system under flow-through conditions were requested by ECHA, for the most critical isomers of the isomeric mixture (1,3-DIPN and 1,4-DIPN) with respect to PBT properties. These two studies are now available and were performed in order to reduce any volatilisation which was identified as a deficiency in the available studies. In the OECD 211 test with 1,3-DIPN the number of alive offspring produced by adults did not show any statistically significant decrease of reproduction up to the highest test concentration of 40 µg/L compared to the solvent control (Eurofins, 2020a). Based on the properties of the test item, the study was performed in a flow-through test design with closed bottles. The measured content of 1,3-DIPN in the replicates was between 42 and 86% of nominal with a mean of 65% of nominal. The measured content of 1,3-DIPN in the fresh stock solutions was between 116 and 136% of nominal and between 117 and 135% of nominal in the aged stock solutions. It is therefore proven that stock solutions remained stable between renewals. The toxicological endpoints were evaluated using nominal test item concentrations and the actual concentrations (based on the time-weighted arithmetic mean of the exposure concentrations). Therefore, the NOEC (21 d) was set to be ≥ 40.0 µg/L (nominal) equivalent to ≥ 24.4 µg/L (measured), including mortality of adults, reproduction and body length.

In the second test with 1,4-DIPN the number of alive offspring produced by adults showed statistically significant decrease of reproduction in the highest test item concentration of 125 µg/L (nominal) equivalent to 38.8 µg/L (measured) compared to the solvent control (Eurofins, 2020b). This study was performed in a flow-through design with closed vessels as well. The measured content of 1,4-Diisopropylnaphthalene was between 16 and 67% of nominal with a mean of 43% of nominal. The measured content of 1,4-Diisopropylnaphthalene was between 95 and 115% of nominal in the fresh stock solutions and between 101 and 119% of nominal in the aged stock solutions. It is therefore proven that stock solutions remained stable between renewals. The toxicological endpoints were evaluated using nominal test item concentrations and the actual concentrations (based on the time-weighted arithmetic mean of the exposure concentrations).

The NOEC (21 d) was calculated to be 62.5 µg/L (nominal) equivalent to 28.1 µg/L (measured) based on reproduction. Statistically significant effects for mortality were observed at 62.5 and 125 µg/L, therefor the NOEC (21 d) for mortality was calculated to be 31.3 µg/L (nominal) equivalent to 16.3 µg/L (measured).

The results of 1,3- and 1,4-DIPN above indicates that the toxicity is above the threshold value of the T-criterion. Both results do not result in fulfilling the T-criterion.

 

For hazard and risk-assessment the NOEC of 0.0118 mg/L was used, which was determined for the long-term toxicity of the isomeric mixture to Daphnids.