2,2'-(cyclohexylimino)bisethanol

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-08-31 to 2011-09-21

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-(cyclohexylimino)bisethanol

EC Number:

224-809-5

EC Name:

2,2'-(cyclohexylimino)bisethanol

Cas Number:

4500-29-2

Molecular formula:

C10H21NO2

IUPAC Name:

2,2'-(cyclohexylimino)bisethanol

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 6.40 - 16.0 - 40.0 - 100 - 250 mg/L The active ingredient 2,2’-(Cyclohexylimino)diethanol was analytically verified by LC-MS/MS in the fresh media (0 h) and old media (48 h) of all concentration levels, the pH-control and the control.
- Sampling method: For the old media additionally replicates with test media but without daphnids and algae were prepared and incubated under test conditions until sampling. All samples were stabilised by diluting 1:2 with methanol directly after sampling.
- Sample storage conditions before analysis: All samples were stored at room temperature until start of analysis.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 250 mg/L was freshly prepared with dilution water.
- Eluate: Dilution water
- Differential loading: 6.40 - 16.0 - 40.0 - 100 - 250 mg/L
- Controls: 10 replicates of dilution water without test item
- pH – CONTROL Additionally, 10 replicates of the highest test concentration were adjusted to a pH in the range 7.0  0.2 by addition of 1 M HCl and tested under the same conditions as the test and control groups

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna STRAUS
- Strain/clone: Clone 5
- Justification for species other than prescribed by test guideline: Daphnia magna STRAUS is recommended in the guideline
- Source: Own breeding (Origin: Institut fuer Wasser- Boden- und Lufthygiene)
- Age of parental stock (mean and range, SD): > 14 days
- Feeding during test
- Food type: Mix of Pseudokirchneriella subcapitata and Desmodesmus subspicatus
- Amount: 0.2 mg C/daphnia per day
- Frequency: daily


ACCLIMATION
- Acclimation period: 2 h in dilution water
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: During acclimation the daphnids were not fed
- Feeding frequency: None
- Health during acclimation (any mortality observed): Healthy

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: The parent animals were removed from the culture medium and the juveniles collected over a sieve and flushed into fresh medium

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

Not observed

Test conditions

Hardness:

Total Hardness [mg/L] as CaCO3
Nominal
Test Item
Concentration
[mg/L] I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 14 Day 16
Aug. 31st Sept. 2nd Sept. 7th Sept. 9th Sept. 14th Sept. 16th
pH-control (250)1) 141 141 155 146 n.d. n.d.
250 146 144 144 144 n.d. n.d.
100 -- -- -- -- 148 150
Control 146 146 141 171 151 142

Test temperature:

Temperature [°C]
Nominal
Test Item
Concentration
[mg/L] I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 14 Day 16
Aug. 31st Sept. 2nd Sept. 7th Sept. 9th Sept. 14th Sept. 16th
pH-control (250)1) 21.3 21.4 20.6 21.7 n.d. n.d.
250 21.3 21.4 20.6 21.4 n.d. n.d.
100 -- -- -- -- 20.9 20.6
Control 21.5 21.0 20.2 21.1 20.8 20.4

pH:

pH-Values
Nominal
Test Item
Concentration
[mg/L] I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 14 Day 16
Aug. 31st Sept. 2nd Sept. 7th Sept. 9th Sept. 14th Sept. 16th
pH-control (250)1) 6.84 7.73 6.97 7.72 n.d. n.d.
250 9.68 8.28 9.62 8.23 n.d. n.d.
100 -- -- -- -- 9.46 8.17
Control 8.46 7.68 8.50 7.75 8.53 7.86

Dissolved oxygen:

Dissolved Oxygen Concentration [mg/L]
Nominal
Test Item
Concentration
[mg/L] I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 14 Day 16
Aug. 31st Sept. 2nd Sept. 7th Sept. 9th Sept. 14th Sept. 16th
pH-control (250)1) 9.07 10.48 8.95 9.15 n.d. n.d.
250 9.06 8.89 8.96 8.90 n.d. n.d.
100 -- -- -- -- 8.92 9.43
Control 9.54 9.25 9.44 8.72 9.35 8.32

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Please refer to Any other information on materials and methods incl. tables

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed with parafilm
- Material, size, headspace, fill volume: 100 mL glass beaker, filled with 50 mL test solution
- Aeration: No
- Renewal rate of test solution (frequency): 3 x per week
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control and pH-control (replicates): 10


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4, according to ELENDT (1990), modified to a total hardness of > 140 mg CaCO3/L.


OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h light : 8 h dark per day
- Light intensity: Max. 20 µE*m-2*s-1


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Adult mortality: daily
- Number of juveniles: daily
- Stillborn juveniles and aborted eggs: daily
- Appearance of first brood
- Intrinsic rate of natural increase: test end
- Growth (total length and dry weight): test end


RANGE-FINDING STUDY
- Test concentrations: No range-finding study

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

46.4 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Remarks on result:

other: (42-51)

Effect concentrations 2

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

20.9 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Remarks on result:

other: (17-25 mg/L)

Effect concentrations 3

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

13 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Remarks on result:

other: n.a.

Effect concentrations 4

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

34.4 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks on result:

other: n.a.

Details on results:

- Mortality of parent animals: The test item induced biologically substantial adult mortality of 70 % in the nominal test item concentration of 100 mg/L and 100 % in the concentration level of 250 mg/L and in the pH-control after 21 days. According to the guideline adult mortality of > 20 % is seen to be biologically substantial. In the concentration levels of 6.40 to 40.0 mg/L and in the control all parental daphnids survived until the end of the study.
The EC10 for the adult mortality was calculated by sigmoidal dose-response regression to be 73.8 mg/L (95% confidence limits: 73.7 – 73.8 mg/L. The EC50 was calculated to be 91.9 mg/L (95 % confidence limits: 40.0 – 250 mg/L), respectively.
- No. of offspring produced per day per female:
Five broods were observed in the test period at all parent animals of the control and the concentration levels 6.40 to 40.0 mg/L. In the concentration level 100 mg/L only 2 of 3 surviving daphnids released broods. There was one parental daphnid releasing two broods and one parental daphnid releasing 3 broods. In the concentration level 250 mg/L and in the pH-control no broods were observed due to 100 % mortality of the daphnids. The average number of juveniles per parent in the control group was 131 after 21 days. The reproductive output was statistically significant reduced in comparison to the control at the nominal test item concentrations of 40.0 and 100 mg/L. The other concentration levels of 6.40 and 16.0 mg/L were not statistically significant reduced when compared to the control (KRUSKAL-WALLIS One Way Analysis of Variance on Ranks, DUNN’S method, p < 0.05). No reproductive output was observed in the concentration level 250 mg/L and in the pH-control (containing 250 mg test item/L) due to 100 % mortality of the daphnids.
- Body length and weight of parent animals: please refer to the respective table
- Type and number of morphological abnormalities: Related to the total number of produced juveniles (dead + alive) the percentage of dead juveniles and aborted eggs was ≤ 3 % at the tested concentration levels 6.40 to 100 mg/L. In the control no dead juveniles or aborted eggs were observed. In the concentration level 250 mg/L and in the pH-control (containing 250 mg test item/L) no juveniles were observed due to 100 % mortality of the daphnids.
- Type and number of behavioural abnormalities: No abnormalities
- Time to first brood release or time to hatch: The first day of appearance of juveniles in the replicates producing juveniles of all concentration levels and the control was released till day 9 by all surviving parental daphnids of the control and the nominal test item concentrations of 6.40 to 40.0 mg/L. In the concentration level of 100 mg/L the first brood was released after day 15, which is regarded to be biologically significantly delayed

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: EC50 (24 h) = 1.70 (CI 1.59 - 1.86)

Reported statistics and error estimates:

The NOEC and the LOEC for the reproductive output as the most sensitive effect and for the adult mortality were determined directly from the observation data. Significant deviations were determined in comparison to the control using statistical standard procedures as Normality Test, Equal Variance Test and Analysis of Variance.
For the determination of significant deviations of the reproductive output, the intrinsic rates of natural increase, the total body length, the day of appearance of the first brood and the number of broods KRUSKAL-WALLIS, One Way Analysis of Variance on Ranks (p < 0.05) was used. The coefficients of variation around the mean number of living offspring produced per parent in the control and the test groups were evaluated.

Any other information on results incl. tables

Number of Juveniles in the Control and Test Groups after 21 Days

Nominal Test Item Conc. [mg/L]												No. of surviving Daphnids	Mean No. of Juveniles per Parent alive			Comparison versus Control
	Number of Juveniles in Replicate No.										Total No.				CV
																Red.	Stat.
	1	2	3	4	5	6	7	8	9	10	å	N	MV ± SD		[%]	[%]
pH-control  (250)^1)	--	--	--	--	--	--	--	--	--	--	--	0	--		--	--	--
250	--	--	--	--	--	--	--	--	--	--	--	0	--		--	--	--
100	--	--	--	--	33	27	0	--	--	--	60	3	20 ±	18	88	85	yes
40.0	111	99	85	82	76	101	87	91	92	91	915	10	92 ±	10	11	30	yes
16.0	121	130	120	120	131	131	134	119	115	135	1256	10	126 ±	7	6	4	no
6.40	122	126	127	130	127	136	123	128	131	130	1280	10	128 ±	4	3	2	no
Control	128	131	126	132	131	132	137	126	135	132	1310	10	131 ±	4	3	—	—

Stillborn Juveniles and Aborted Eggs after 21 Days

Nominal Test Item Concentration	Number of		Total No.		Mean Stillborn Juveniles and Aborted Eggs per Parent alive
	Stillborn	Aborted
[mg/L]	Juveniles	Eggs	å	N	MV ± SD
pH-control (250)^1)	--	--	--	--	--
250	--	--	--	--	--
100	0	2	2	3	0.67 ± 1.15
40.0	21	10	31	10	3.10 ± 1.91
16.0	22	7	29	10	2.90 ± 2.13
6.40	2	5	7	10	0.70 ± 1.06
Control	0	0	0	10	0 ± 0

First Appearance of Living Juveniles in the Individual Groups

Nominal Test Item Concentration	Day of First Appearance of Living Juveniles										First
	in Replicate No.										Appearance
[mg/L]	1	2	3	4	5	6	7	8	9	10	Mean Day
pH-control (250)^1)	--	--	--	--	--	--	--	--	--	--	--
250	--	--	--	--	--	--	--	--	--	--	--
100	--	--	--	--	15^2)	19^2)	n.a.	--	--	--	17.0^2)
40.0	9	8	8	8	8	8	8	8	9	8	8.2
16.0	9	8	8	8	8	8	9	8	8	8	8.2
6.40	8	8	9	8	8	8	8	8	8	8	8.1
Control	9	8	8	8	8	8	8	8	9	8	8.2

Mortality [%] of the Adult Daphnids after 7, 14 and 21 Days of Exposure

                 (n = 10)

Nominal Test Item Concentration	Adult Mortality [%]
[mg/L]	7 days	14 days	21 days
pH-control (250)^1)	0	100^2)	100^2)
250	80^2)	100^2)	100^2)
100	0	10	70^2)
40.0	0	0	0
16.0	0	0	0
6.40	0	0	0
Control	0	0	0

Total Body Length and Dry weight of the Surviving Parental Daphnids

Nominal Test Item Concentration [mg/L]	Total Length of the Parent Animals [mm]										MV	N	Dry Weight [mg]
	Replicate No.
	1	2	3	4	5	6	7	8	9	10	[mm]		S	MV
pH-control (250)^1)	--	--	--	--	--	--	--	--	--	--	--	0	--	--
250	--	--	--	--	--	--	--	--	--	--	--	0	--	--
100	--	--	--	--	4.50	4.75	3.75	--	--	--	4.33	3	0.1	0.03
40.0	5.50	5.75	5.50	5.25	5.25	5.50	5.75	5.75	5.25	5.50	5.50	10	7.1	0.71
16.0	5.75	5.50	5.50	5.50	5.50	5.25	5.50	5.25	5.50	5.75	5.50	10	5.9	0.59
6.40	5.00	5.25	5.25	5.50	5.25	5.50	5.00	5.50	5.50	5.25	5.30	10	6.7	0.67
Control	5.00	5.25	5.25	5.50	5.50	5.50	5.00	5.25	5.00	5.25	5.25	10	6.6	0.66

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The Lowest Observed Effect Concentration (LOEC) and the No Observed Effect Concentration (NOEC) for the reproductive output as the most sensitive effect and the adult mortality were determined directly from the observation data.
The EC10- and EC50-values for the reproductive output and the adult mortality were calculated by sigmodial dose-response regression. All effect values given are based on the nominal concentrations of the test item 2,2’-(Cyclohexylimino)diethanol.

EC10, Reproduction : 24.3 mg/L (95 % confidence limits: 20.3 – 29.1 mg/L)
EC50, Reproduction : 54.0 mg/L (95 % confidence limits: 49.1 – 59.7 mg/L)
NOECReproduction : 16.0 mg/L
LOECReproduction : 40.0 mg/L

EC10, Adult Mortality : 73.8 mg/L (95 % confidence limits: 73.7 – 73.8 mg/L)
EC50, Adult Mortality : 91.9 mg/L (95 % confidence limits: 40.0 – 250 mg/L)
EC100, Adult Mortality : 250 mg/L
NOECAdult Mortality : 40.0 mg/L
LOECAdult Mortality : 100 mg/L

Executive summary:

The Daphnia magna Reproduction Test (semi-static, 21 d) of the test item 2,2’-(Cyclohexylimino)diethanol(batch number ESD0011452) was conducted according to OECD 211 (2008) from 2011-08-31 to 2011-09-22, with the definitive exposure phase from 2011-08-31 to 2011-09-21, at Dr.U.Noack-Laboratorien, Käthe-Paulus-Str. 1, 31157Sarstedt, Germany.

Test species was Daphnia magna STRAUS (Clone 5). Ten daphnids, individually held, were used per concentration level, pH-control and control. At test start the daphnids were 2 to 24 hours old. The study was carried out under semi-static conditions with renewal of the test solutions 3 times per week. Aim of the Daphnia Reproduction Test over 21 days was to assess effects on the reproduction capacity and other test item-related effects or parameters such as time of production of first brood, adult mortality, intrinsic rate of natural increase, occurrence of aborted eggs and stillborn juveniles and body length of the parental daphnids.

Nominal concentrations of 2,2’-(Cyclohexylimino)diethanolwere selected as follows: 6.40 - 16.0 - 40.0 - 100 - 250 mg/L.

The concentrations of the test item 2,2’-(Cyclohexylimino)diethanol were analytically verified by LC-MS/MS of samples taken on days 0, 7, 14 (fresh media, 0 hours) and on days 2, 9, 16 (old media, 48 hours) of all concentration levels, the pH-control and the control.

The recoveries in the fresh media (0 hours) and in the old media (48 hours) were in the range of 92 to 106 %, demonstrating that concentrations of 2,2’-(Cyclohexylimino)diethanolwere successfully maintained for the duration of the test. Therefore, all effect values given are based on the nominal concentrations of the test item 2,2’-(Cyclohexylimino)diethanol.

·   The average number of juveniles per parent in the control group was 131 after 21 days. The reproductive output was statistically significant reduced in comparison to the control at the nominal test item concentrations of 40.0 and 100 mg/L. The other concentration levels of 6.40 and 16.0 mg/L were not statistically significant reduced when compared to the control (Kruskal-Wallis One Way Analysis of Variance on Ranks, Dunn’smethod, p  0.05). No reproductive output was observed in the concentration level 250 mg/L and in the pH-control (containing 250 mg test item/L) due to 100 % mortality of the daphnids.
The EC₁₀ for the reduction of the reproductive output was calculated by sigmoidal dose-response regression to be 24.3 mg/L (95% confidence limits: 20.3 – 29.1 mg/L). The EC₅₀ was calculated to be 54.0 mg/L (95 % confidence limits: 49.1 – 59.7 mg/L), respectively.

·   The coefficient of variation of the number of living offspring produced per parent was 3 % in the control.

·   The Lowest Observed Effect Concentration (LOEC_Reproduction) and the No Observed Effect Concentration (NOEC_Reproduction) after 21 days based on the reproductive output as the most sensitive effect are summarized in Table 1.

The intrinsic rates of natural increase (IR)of the surviving parent animals accounting for generation time and number of offspring were used for calculation of population growth and maintenance. The mean IR of the surviving daphnids of the treatment groups were compared to the control by Kruskal-WallisOne Way Analysis of Variance on Ranks, Dunn’smethod (p  0.05). There was a statistically significant difference at the concentration levels of 40.0 and 100 mg/L. No intrinsic rate could be calculated for the concentration level 250 mg/L and the pH-control (containing 250 mg test item/L) due to 100 % mortality of the daphnids. The other concentration levels of 6.40 and 16.0 mg/L were not statistically significant when compared to the control.

·   Related to the total number of produced juveniles (dead + alive) the percentage of dead juveniles and aborted eggs was ≤ 3 % at the tested concentration levels 6.40 to 100 mg/L (Table 7). In the control no dead juveniles or aborted eggs were observed. In the concentration level 250 mg/L and in the pH-control (containing 250 mg test item/L) no juveniles were observed due to 100 % mortality of the daphnids.

·   Five broods were observed during the test period at all surviving parental daphnids in the concentration levels of 6.40 to 40.0 mg/L. In the concentration level 100 mg/L the number of broods was significantly reduced to ≤ 3 broods per surviving daphnid during the exposure phase (Kruskal-WallisOne Way Analysis of Variance on Ranks, Dunn’smethod, p  0.05). In the concentration level 250 mg/L and in the pH-control no broods were observed due to 100 % mortality of the daphnids. The first brood was released till day 9 by all surviving parental daphnids of the control and the nominal test item concentrations of 6.40 to 40.0 mg/L. In the concentration level of 100 mg/L the first brood was released after day 15, which is regarded to be biologically significantly delayed.

Test Item Related Effects on Reproduction (EC₁₀, EC₅₀, NOEC and LOEC)

Effects	2,2’-(Cyclohexylimino)diethanol Nominal Test Item Concentration [mg/L]
	Control	6.40	16.0	40.0	100	250	pH-control^1)
Mean Number of Juveniles per Producing Parental Daphnid (Reproduction Rate ± SD)	131 ± 4	128 ± 4	126 ± 7	92 ± 10^2)	20 ± 18^2)	--	--
Mean Intrinsic Rates of Natural Increase	0.50	0.50	0.49	0.44^2)	0.15^2)	--	--
Appearance of First Brood [Mean Day]	8.2	8.1	8.2	8.2	17.0^3)	--	--
EC10, Reproduction (with 95 % confidence limits)	24.3 mg(L (Cl: 20.3 – 29.1 mg/L)
EC50, Reproduction (with 95 % confidence limits)	54.0 mg/L (Cl: 49.1 – 59.7 mg/L)
NOECReproduction	16.0 mg/L
LOECReproduction	40.0 mg/L

A summary of test item related effects concerning the adult mortality (EC_{10, Adult Mortality},         EC_{50, Adult Mortality}, NOEC_{Adult Mortality} and LOEC_{Adult Mortality}) and growth (dry weight, body length) is given in Table 2.

·   The test item induced biologically substantial adult mortality of 70 % in the nominal test item concentration of 100 mg/L and 100 % in the concentration level of 250 mg/L and in the pH-control after 21 days. According to the guideline adult mortality of > 20 % is seen to be biologically substantial. Inthe concentration levels of 6.40 to 40.0 mg/L and in the control all parental daphnids survived until the end of the study.
The EC₁₀ for the adult mortality was calculated by sigmoidal dose-response regression to be 73.8 mg/L (95% confidence limits: 73.7 – 73.8 mg/L. The EC₅₀ was calculated to be 91.9 mg/L (95 % confidence limits: 40.0 – 250 mg/L), respectively.

·   The mean body lengths of the surviving parental daphnids in the nominal test item concentrations 6.40 to 100 mg/L were determined to be in the range of 4.33 to 5.50 mm per daphnid and 5.25 mm per daphnid in the control group, respectively (Table 2 and Table 10). The body length of the surviving daphnids was not statistically significantly reduced at any of the tested concentration levels in comparison to the control (Kruskal-Wallis One Way Analysis of Variance on Ranks, Dunn’smethod, p  0.05).
The mean dry body weight was determined to be in the range of 0.03 to 0.71 mg per daphnid in the test groups and 0.66 mg per daphnid in the control, respectively.

Table 2:    Test Item Related Effects on Adult Mortality (EC₁₀, EC₅₀, NOEC and LOEC) and Growth

Effects	2,2’-(Cyclohexylimino)diethanol Nominal Test Item Concentration [mg/L]
	Control	6.40	16.0	40.0	100	250	pH-control^1)
Adult mortality after 21 days [%]	0	0	0	0	70^2)	100^2)	100^2)
Mean body length of parental daphnids [mm]	5.25	5.30	5.50	5.50	4.33	--	--
Dry weight of parental daphnids [mg]	0.66	0.67	0.59	0.71	0.03	--	--
EC10, Adult Mortality (with 95 % confidence limits)	73.8 mg(L (Cl: 73.7 – 73.8 mg/L)
EC50, Adult Mortality (with 95 % confidence limits)	91.9 mg/L (Cl: 40.0 – 250 mg/L)
EC100, Adult Mortality	250 mg/L
NOECAdult Mortality	40.0 mg/L
LOECAdult Mortality	100 mg/L

·       No males and ephippia (winter eggs) occurred in control or test groups.

 

·   Water quality parameters as pH-value, dissolved oxygen, water hardness and temperature were determined to be within the acceptable limits.

·   In order to prove the validity of the test system and test conditions at the test facility, an acute immobilization test according to DIN 38412 L 11 was carried out with potassium dichromate as reference item once per month.
The EC₅₀ of the reference item at 1.70 mg/L after 24 hours was within the prescribed concentration range of 1.0 - 2.5 mg/L of quality criteria according to AQS P 9/2 (05/1996) for daphnids clone 5 cultured in Elendt M4 medium. The EC₅₀-value of the reference item is also within the recommended range of 0.6 - 2.1 mg/L according to OECD-Guideline 202.