2-(2,4-diaminophenoxy)ethanol dihydrochloride

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2016-08-25 to 2017-06-22

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)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I74820
- Expiration date of the lot/batch: 2017-03-22
- Purity test date: 2014-03-26

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, protected from light, in an evacuated desiccator, in the tightly closed original container
- Stability under test conditions: not specified
- Solubility and stability of the test substance in the solvent/vehicle: in water: soluble
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: nd

TREATMENT OF TEST MATERIAL PRIOR TO TESTING no

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.500 - 1.00 - 2.00 - 4.00 - 8.00 mg/L,
- Sampling method: At the start of the exposure intervals, samples of the fresh media were taken after preparation of all test item concentrations and analyzed.
At the end of the exposure intervals (48 or 72 hours), samples of the old media were taken directly from the test vessels.For the longest exposure interval of 72 hours samples were taken at the start (0 hours) and at the end of the exposure interval (72 hours) once within the test period.
- Sample storage conditions before analysis: All samples were analyzed on the day of collection keeping storage time as low as possible. The samples were stored until analysis in an auto-sampler at room temperature. Additionally, the samples were stabilized by addition of acetonitrile (dilution factor 2) directly after sampling to prevent degradation during the storage.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
A stock solution (8.00 mg/L of the test item were weighed out) was freshly prepared with dilution water for each exposure interval. The stock solution was mixed thoroughly by manual agitation and was used as highest test concentration and for preparation of the further test concentrations.
- Controls: 10 replicates of dilution water without test item
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): na
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): not specified

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna STRAUS
- Strain/clone: Clone 5
- Source: Niedersächsischer Landesbetrieb für Wasserwirtschaft, Küsten- und Naturschutz, An der Scharlake 39, 31135 Hildesheim, Germany
- Age of parental stock (mean and range, SD): bred in Noack Laboratorien
- Feeding during test: Yes
- Food type/amount/frequency: Daily feeding per test vessel; Pseudokirchneriella subcapitata (0.889 - 1.35 mL) and Desmodesmus subspicatus (0.650 - 0.877 mL) suspension was provided as food corresponding to 0.2 mg C per Daphnia and day. There was variation according to the density of the algae suspension, but it was the same for all test groups on each feeding day.

ACCLIMATION: no acclimation necessary since test dilution water is the same as culture medium

QUARANTINE (wild caught) na

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: Less than 24 hours old daphnids from a healthy stock were used for the study. Juvenile daphnids were removed from the culture vessels at the latest 24 hours before the start of the exposure and discarded. The juveniles born within this period of max. 24 hours preceding the exposure were used for the test. No first brood progeny was used for the test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

The total hardness was above 140 mg/L as CaCO3 during the whole study period.

Test temperature:

The temperature was within the range of 20 ± 1 °C during the whole study period.

pH:

Variations between 7.06-7.2 and 8.63-7.84. Significant deviations ≥ 1.5 units between the initial and final pH-values were not observed.

Dissolved oxygen:

The dissolved oxygen concentration was above 3 mg/L during the whole study period.

Nominal and measured concentrations:

Nominal : 0.500 - 1.00 - 2.00 - 4.00 - 8.00 mg/L
Geometric mean measured concentrations : 0.430 - 0.853 - 1.68 - 3.48 - 6.46 mg/L.
The measured concentrations verified by LC-MS/MS in fresh media at the start of the respective exposure intervals (0 hours) were in the range of 81% to 119% of the nominal concentrations. At the end of the respective exposure intervals (48 and 72 hours), the measured test item concentrations in the old media were in the range of 54 to 85% of the nominal concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel/Material, size, headspace, fill volume: Glass beakers (5 (ID) x 8 (H) cm), 100 mL capacity; test volume: 50 mL
- Type (delete if not applicable):closed; loosely covered with watch glasses
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): na
- Renewal rate of test solution (frequency/flow rate): The test solutions were renewed 3 times per week (after 48 or 72 hours). For this purpose, a second set of test vessels was filled with the freshly prepared test solutions and the daphnids were transferred by pipette.
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- No. of vessels per vehicle control (replicates): na
- Biomass loading rate: 1 daphnia/50 mL

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4, according to OECD 202, Annex 3 (2004), modified to a total hardness of 160 to 180 mg CaCO3/L, is used.
- Culture medium different from test medium: no
- Intervals of water quality measurement: The water quality parameters (i.e. pH-values, dissolved oxygen concentration, total hardness and water temperature) were measured once within 7 days, in fresh media at the start (0 hours) and in old media at the end (48 or 72 hours) of an exposure interval, in one replicate of the control and the highest test concentration. Since 100% mortality was observed in the concentration level 8.00 mg/L at test day 5, the measurements were carried out in the concentration level 8.00 mg/L only for the first exposure interval and thereafter in the following concentration level 4.00 mg/L for the remaining exposure intervals. The water quality parameters in fresh media at the start of an exposure interval were measured in an additional replicate without daphnids of the highest test concentration and the control. At the end of an exposure interval, the water quality parameters of the old media were measured in a test vessel of the highest test concentration and the control, which contained daphnids and food algae. The temperature in the incubator was recorded throughout the test period with a thermo-hygrograph

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16/8 hours light/dark cycle
- Light intensity: Max. 1500 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : effects on the reproduction capacity and other test item-related effects or parameters such as the intrinsic rate of the natural increase, first appearance of living juveniles, occurrence of aborted eggs and stillborn juveniles, adult mortality, body length and dry weight of the parental daphnids.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
- Justification for using less concentrations than requested by guideline: na
- Range finding study: the acute daphnia study (according to OECD 202 and GLP) was served as range finding study

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.43 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

0.853 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

0.35 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: CI: 0.322-0.380

Key result

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

1.35 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: CI: 1.22-1.49

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1.68 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

3.48 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

21 d

Dose descriptor:

other: LC20

Effect conc.:

1.17 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

21 d

Dose descriptor:

LC50

Effect conc.:

2.75 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

21 d

Dose descriptor:

other: LC100

Effect conc.:

6.46 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

- Behavioural abnormalities: none
- Observations on body length and weight: The mean values of the body length (excluding the anal spine) of the survived parental daphnids in the tested concentration levels 0.430 to 3.48 mg/L were in the range of 4.40 to 4.91 mm per daphnid and 4.88 mm per daphnid in the control group. The mean dry weight of the survived parental daphnids was in the range of 0.62 to 0.86 mg per daphnid in the concentration levels 0.430 to 3.48 mg/L and 0.67 mg per daphnid in the control.
- Other biological observations:
Reproductive Output: The average number of living juveniles at the end of the test after 21 days per survived parental daphnid was 113 in the control group and thus the validity criterion for the reproduction of a mean of ≥ 60 living offspring per survived parent animal in the control was met.
No parental daphnids were excluded due to accidental or inadvertent mortality from the evaluation of the reproductive output. Since the Cochran-Armitage test procedure revealed a significant increasing trend (p ≤ 0.05) in adult mortality, the evaluation was done based on the cumulative offspring per introduced parent. A statistically significant reduction of the reproductive output in comparison to the reproductive output in the control was determined at the concentration levels 0.853 to 6.46 mg/L (Williams multiple sequential t-test procedure, α = 0.05). The determination of the EC10- and the EC50-value and their 95% confidence limits was carried out by point estimates from the 3-parametric normal CDF.
The parameter living offspring per survived parental animal at the end of exposure was also evaluated and the results were comparable to the cumulative offspring per introduced parent animal. Nevertheless, the evaluation based on the cumulative offspring per introduced parent is the ecologically most relevant response variable.
Adult Mortality: Accidental and inadvertent mortality as defined in the OECD test guideline were not observed.
The test item induced statistically significant adult mortality of 100% in the concentration level 6.46 mg/L and 50% in the concentration level 3.48 mg/L (Step down Cochran-Armitage Test Procedure, α = 0.05). In the concentration level 0.430 mg/L, a mortality rate of 20% of the introduced adults was observed as well as 10% mortality in the concentration level 1.68 mg/L, which is not statistically significant. In the concentration level of 0.853 mg/L and in the control all parental daphnids survived until the end of the test (21 days). The Cochran-Armitage test procedure revealed a significant increasing trend (p ≤ 0.05) in adult mortality. Therefore, the LC20- and LC50-values were calculated by probit analysis.
The intrinsic rates of natural increase (IR) of the survived parental daphnids accounting for generation time and number of offspring were used for calculation of population growth and maintenance.
The first appearance of living juveniles was observed between days 7 and 12 at all introduced parental daphnids of the control and the concentration levels 0.430 to 3.48 mg/L. In the highest concentration level 6.46 mg/L, all parental daphnids died during the exposure period.
No stillborn juveniles or aborted eggs were observed in the control and in the concentration level 0.430 mg/L during the exposure period of 21 days. Related to the total number of produced juveniles (dead + alive) per survived daphnid, the percentage of dead juveniles was ≤ 60% in the concentration levels 0.853 to 3.48 mg/L.
No males were observed in the control or in the test groups during the test.
No ephippia were observed in the control or in the test groups during the test.
- Mortality of control: 0% at the end of the 21d exposure
- Other adverse effects control: none
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none
- Effect concentrations exceeding solubility of substance in test medium: none

Results with reference substance (positive control):

- Results with reference substance valid? Yes
The percentage of immobility for the reference item potassium dichromate (SIGMA-ALDRICH, batch number MKBV0900V, purity 99.0%, CAS RN 7778-50-9) was determined after 24 hours from 2016 11-24 to 2016-11-25.

Current Study: 24H-EC50=0.802 mg/L (95% CI: 0.597 - 0.942 mg/L)
Valid Range: 24H-EC50=0.6 - 2.1 mg/L, acc. to OECD 202 (2004); clone A

Reported statistics and error estimates:

See above

Mortality [%] of the Adult Daphnids after 7, 14 and 21 Days of Exposure (n = 10)

Geometric mean measured test item concentrations [mg/L]	Adult Mortality [%]
	7 days	14 days	21 days
6.46	100	100	100^1)
3.48	30	40	50^1)
1.68	0	0	10
0.853	0	0	0
0.430	0	0	20
Control	0	0	0

1)    = statistically significant (Step‑down Cochran-Armitage Test Procedure,α = 0.05)

 

Intrinsic Rates of Natural Increase

Geometric mean measured test item concentrations [mg/L]	Intrinsic rate of natural increase in replicate no.										Mean IR		CV
	1	2	3	4	5	6	7	8	9	10	MV±SD		[%]
6.46	--	--	--	--	--	--	--	--	--	--	--		--
3.48	--	--	--	0.101	--	0.101	0.101	--	0.101	0.248	0.130±	0.0655	50.2
1.68	0.339	0.390	0.396	0.366	0.461	0.366	0.446	0.363	0.443	--	0.397±	0.0435	11.0
0.853	0.316	0.312	0.432	0.440	0.408	0.305	0.319	0.314	0.369	0.421	0.364±	0.0564	15.5
0.430	--	0.413	0.367	0.426	--	0.374	0.405	0.401	0.439	0.414	0.405±	0.0244	6.0
Control	0.454	0.365	0.441	0.363	0.341	0.471	0.427	0.344	0.388	0.374	0.397±	0.0474	11.9

--  = not applicable, due to the mortality of the parental daphnid(s)

 

First Appearance of Living Juveniles in the Individual Groups

Geometric mean measured test item concentrations [mg/L]	Day of first appearance of living juveniles at the introduced parentaldaphnidsin replicate no.										First
											appearance
	1	2	3	4	5	6	7	8	9	10	mean day
6.46	--	--	--	--	--	--	--	--	--	--	--
3.48	--	--	--	no	10	no	12	--	9	10	10.3
1.68	9	9	9	9	7	9	9	9	9	8	8.7
0.853	10	9	9	9	9	9	10	10	9	9	9.3
0.430	9	10	9	9	9	8	8	9	8	8	8.8
Control	8	9	9	9	10	7	9	10	9	9	8.9

--  = not applicable, due to the mortality of the parental daphnid(s)

*   = only broods of the survived parental daphnids with living juveniles taken into account

no       = no broods occurred

 

 Stillborn Juveniles and Aborted Eggs produced by the Survived Parental daphnids after 21 Days

Geometric mean measured test item concentrations [mg/L]	Number of		Total no. Ʃ	Number of survived adults N
	stillborn juveniles	aborted eggs
6.46	--	--	--	0
3.48	37	37	74	5
1.68	43	55	98	9
0.853	29	27	56	10
0.430	0	0	0	8
Control	0	0	0	10

N  = number of the survived parental daphnids

--  = not applicable, due to the mortality of the parental daphnids

 

 Relative Number of Dead (Stillborn + Aborted Eggs) to Total Number of Juveniles per Survived Parental daphnids

Geometric mean measured test item concentrations [mg/L]	Number of juveniles			Percentage of dead juveniles^#) [%]
	Dead	Alive	Total
6.46	--	--	--	--
3.48	74	49	123	60
1.68	98	682	780	13
0.853	56	498	554	10
0.430	0	887	887	0
Control	0	1133	1133	0

Dead = aborted eggs + stillborn juveniles

Total = dead + alive juveniles

^#)       = related to the total number of juveniles

--      = not applicable, due to the mortality of the parental daphnid(s)

 

Total Body Length and Dry Weight of the Survived Parental daphnids

Geometric mean measured test item concentrations [mg/L]	Total length of the survived parent animals [mm]										MV	N	Dry weight [mg]
	Replicate no.
	1	2	3	4	5	6	7	8	9	10	[mm]		Ʃ	MV
6.46	--	--	--	--	--	--	--	--	--	--	--	0	--
3.48	--	--	--	3.50	--	4.50	4.75	--	4.50	4.75	4.40	5	3.1	0.62
1.68	4.50	5.00	5.00	5.00	4.75	4.50	4.75	4.50	4.75	--	4.75	9	7.7	0.86
0.853	4.75	5.00	5.00	4.75	5.00	4.50	5.00	4.75	4.50	4.75	4.80	10	8.4	0.84
0.430	--	5.00	5.00	5.00	--	5.00	4.75	4.75	4.75	5.00	4.91	8	6.1	0.76
Control	5.00	4.75	5.00	5.00	4.75	4.75	5.00	5.00	4.75	4.75	4.88	10	6.7	0.67

N = number of the survived parental daphnids

--  = not applicable, due to the mortality of the parental daphnid(s)

 

 

Validity criteria fulfilled:

yes

Remarks:

Mortality of the parent in the controls did not exceed 20% at the end of the test. The mean number of living offspring produced per survived parent at the end of the test was 113 in the controls (required: at least 60 juveniles per parental daphnid).

Conclusions:

The effects on reproduction were evaluated based on the reproduction per introduced parent animal. The assessed effects of the test item under test conditions are all based on the geometic mean measured concentrations.
EC10 Reproduction= 0.350 mg/L; EC50 Reproduction=1.35 mg/L; LOEC Reproduction=0.853 mg/L; NOEC Reproduction =0.430 mg/L; LC20 Adult mortality after 21 days=1.17 mg/L; LC50 Adult mortality after 21 days=2.75 mg/L; LC100 Adult mortality after 21 days=6.46 mg/L; LOEC Adult mortality after 21 days=3.48 mg/L; NOEC Adult mortality after 21 days=1.68 mg/L
All effect levels (NOEC, LOEC, EC10 / 50 / 100 and LC20 / 50 / 100) given are based on the geometric mean measured test item concentrations, since the measured test item concentrations were not within ± 20% of the nominal concentrations.

Executive summary:

The purpose of the study was to determine the long term effect over 21 days of the test item on Daphnia magna, according to the OECD 211 guideline and following GLP. Ten daphnids, held individually, were used per concentration level and control. At test start the daphnids were 2 to 24 hours old. The study was carried out under semi-static conditions with a medium renewal three times per week. Aim of the test was to assess the effects on the reproduction capacity and other test item-related effects or parameters such as the intrinsic rate of the natural increase, first appearance of living juveniles, occurrence of aborted eggs and stillborn juveniles, adult mortality, body length and dry weight of the parental daphnids. Nominal concentrations of the test item were selected based on the results of an acute immobilization test as follows: 0.500 - 1.00 - 2.00 - 4.00 - 8.00 mg/L. The test item concentrations were analytically verified via LC-MS/MS in fresh media at the start of the exposure intervals on days 0, 12, 16 (0 hours) and in old media at the end of the exposure intervals on days 2, 14, 19 (48 or 72 hours, respectively) in all concentration levels with surviving parental daphnids and the control. The measured concentrations of the test item in fresh media at the start of the respective exposure intervals (0 hours) were in the range of 81 to 119% of the nominal concentrations. At the end of the respective exposure intervals (48 and 72 hours), the measured test item concentrations in the old media were in the range of 54 to 85% of the nominal concentrations.

The geometric mean measured concentrations are: 0.430 - 0.853 - 1.68 - 3.48 - 6.46 mg/L.

The environmental conditions were within the acceptable limits. The validity criteria of the OECD 211 test guideline were met. The effects on reproduction were evaluated based on the reproduction per introduced parent animal.

EC10 Reproduction= 0.350 mg/L; EC50 Reproduction=1.35 mg/L; LOEC Reproduction=0.853 mg/L; NOEC Reproduction =0.430 mg/L; LC20 Adult mortality after 21 days=1.17 mg/L; LC50 Adult mortality after 21 days=2.75 mg/L; LC100 Adult mortality after 21 days=6.46 mg/L; LOEC Adult mortality after 21 days=3.48 mg/L; NOEC Adult mortality after 21 days=1.68 mg/L

Description of key information

The effects on reproduction were evaluated based on the reproduction per introduced parent animal. The assessed effects of the test item under test conditions are all based on the geometic mean measured concentrations.

EC10 Reproduction= 0.350 mg/L; EC50 Reproduction=1.35 mg/L; LOEC Reproduction=0.853 mg/L; NOEC Reproduction =0.430 mg/L; LC20 Adult mortality after 21 days=1.17 mg/L; LC50 Adult mortality after 21 days=2.75 mg/L; LC100 Adult mortality after 21 days=6.46 mg/L; LOEC Adult mortality after 21 days=3.48 mg/L; NOEC Adult mortality after 21 days=1.68 mg/L

All effect levels (NOEC, LOEC, EC10 / 50 / 100 and LC20 / 50 / 100) given are based on the geometric mean measured test item concentrations, since the measured test item concentrations were not within ± 20% of the nominal concentrations.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.43 mg/L

Additional information

The purpose of the study was to determine the long term effect over 21 days of the test item on Daphnia magna, according to the OECD 211 guideline and following GLP. Ten daphnids, held individually, were used per concentration level and control. At test start the daphnids were 2 to 24 hours old. The study was carried out under semi-static conditions with a medium renewal three times per week. Aim of the test was to assess the effects on the reproduction capacity and other test item-related effects or parameters such as the intrinsic rate of the natural increase, first appearance of living juveniles, occurrence of aborted eggs and stillborn juveniles, adult mortality, body length and dry weight of the parental daphnids. Nominal concentrations of the test item were selected based on the results of an acute immobilization test as follows: 0.500 - 1.00 - 2.00 - 4.00 - 8.00 mg/L. The test item concentrations were analytically verified via LC-MS/MS in fresh media at the start of the exposure intervals on days 0, 12, 16 (0 hours) and in old media at the end of the exposure intervals on days 2, 14, 19 (48 or 72 hours, respectively) in all concentration levels with surviving parental daphnids and the control. The measured concentrations of the test item in fresh media at the start of the respective exposure intervals (0 hours) were in the range of 81 to 119% of the nominal concentrations. At the end of the respective exposure intervals (48 and 72 hours), the measured test item concentrations in the old media were in the range of 54 to 85% of the nominal concentrations.

The geometric mean measured concentrations are: 0.430 - 0.853 - 1.68 - 3.48 - 6.46 mg/L.

The environmental conditions were within the acceptable limits. The validity criteria of the OECD 211 test guideline were met. The effects on reproduction were evaluated based on the reproduction per introduced parent animal.

EC10 Reproduction= 0.350 mg/L; EC50 Reproduction=1.35 mg/L; LOEC Reproduction=0.853 mg/L; NOEC Reproduction =0.430 mg/L; LC20 Adult mortality after 21 days=1.17 mg/L; LC50 Adult mortality after 21 days=2.75 mg/L; LC100 Adult mortality after 21 days=6.46 mg/L; LOEC Adult mortality after 21 days=3.48 mg/L; NOEC Adult mortality after 21 days=1.68 mg/L