4-[(2,5-dichlorophenyl)azo]-N-(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)-3-hydroxynaphthalene-2-carboxamide

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:

2011-11-27 to 2011-12-21

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

according to German Chemikaliengesetzt and directive 88/320/EEC

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 1 mg/L was prepared with dilution water two days prior to application. The stock solution was stirred with approximately 1100 rpm for 48 hours at room temperature. Undissolved particles were removed by membrane filtration (membrane filter: 0.45 µm, RC, MACHEREY-NAGEL). Due to the test item properties centrifugation was not sufficient.
- Eluate: Dilution water
- Differential loading: Saturated solution as limit concentration
- Controls: 10 replicates of dilution water without test item.

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

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

21 d

Post exposure observation period:

Not observed

Hardness:

Total Hardness as CaCO3 [mg/L]
Nominal
Test Item
Concentration
[mg/L] I F I F I F
Day 0 Day 1 Day 8 Day 9 Day 15 Day 16
2011-11-29 2011-11-30 2011-12-07 2011-12-08 2011-12-14 2011-12-15
1.00
(Saturated solution) 173 169 173 176 180 180
Control 176 167 173 171 164 169

Test temperature:

Temperature [°C]
Nominal
Test Item
Concentration
[mg/L] I F I F I F
Day 0 Day 1 Day 8 Day 9 Day 15 Day 16
2011-11-29 2011-11-30 2011-12-07 2011-12-08 2011-12-14 2011-12-15
1.00
(Saturated solution) 20.0 20.5 21.2 20.9 21.0 20.9
Control 19.8 20.2 21.4 20.7 20.5 20.9

pH:

pH-Values
Nominal
Test Item
Concentration
[mg/L] I F I F I F
Day 0 Day 1 Day 8 Day 9 Day 15 Day 16
2011-11-29 2011-11-30 2011-12-07 2011-12-08 2011-12-14 2011-12-15
1.00
(Saturated solution) 8.15 8.38 7.85 7.83 8.06 7.55
Control 8.04 8.02 7.85 7.67 7.85 7.53

Dissolved oxygen:

Dissolved Oxygen Concentration [mg O2/L]
Nominal
Test Item
Concentration
[mg/L] I F I F I F
Day 0 Day 1 Day 8 Day 9 Day 15 Day 16
2011-11-29 2011-11-30 2011-12-07 2011-12-08 2011-12-14 2011-12-15
1.00
(Saturated solution) 5.21 10.13 5.03 9.13 5.25 7.65
Control 9.81 9.53 8.34 8.48 8.62 7.87

Salinity:

Not measured, freshwater

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): daily renewal
- 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
A preliminary test was carried out as acute immobilization test with the saturated solution with 1 mg test item/L under static conditions over a period of 48 hours. No effects (including immobilization, signs of disease or stress, e.g. discoloration or unusual behaviour) were observed on Daphnia magna after an exposure period of 48 hours.
Immobilization Rates in the Preliminary Test
(n = 20, divided into 2 replicates with 10 daphnids each)
Nominal
Test Item Concentration
[mg/L] IMMOBILIZATION [%]
24 hours 48 hours
Replicates Replicates
1 2 MV 1 2 MV
1.00
(Saturated solution) 0 0 0 0 0 0
Control 0 0 0 0 0 0

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

and mortality

Remarks on result:

other: As the test item is highly insoluble in water and solvents, no suitable method for determination of the test item could be established.

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: reproduction and mortality

Remarks on result:

other: As the test item is highly insoluble in water and solvents, no suitable method for determination of the test item could be established.

Details on results:

- Mortality of parent animals:
No significant adult mortality or immobilization of parental daphnids was observed in control and the saturated solution with 1.00 mg test item/L. The adult mortality was 10 % in the control and in the saturated test item solution, respectively.
- No. of offspring produced per day per female:
In the control and the saturated test item solution (limit concentration), five broods were released during the test period by all daphnids, respectively. The first brood was released on days 8 and 9 by all parent daphnids in the control and test group.

The mean number of alive offspring produced per parent daphnid surviving the test was 125 in the control and 126 in the saturated test item solution with 1.00 mg test item/L after 21 days, respectively. The reproductive output of daphnids in the saturated test item solution (limit concentration) was comparable to the output of daphnids in the control (t-test, p = 0.05).
- Body length and weight of parent animals:
Please refer to the respective table
- Type and number of morphological / behavioural abnormalities:
Related to the total number of produced juveniles (dead + alive) the percentage of dead juveniles was = 1 % at the control and the saturated solution, which is seen to be not biologically significant.

- Time to first brood release or time to hatch:
The first brood was released from all parent daphnids in all treatments until day 9

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: EC50 (24 h) = 1.63 (CI 1.60 - 1.66) mg/L

Reported statistics and error estimates:

The NOEC and LOEC for reproduction as measured by the intrinsic rate of natural increase and total living neonates per surviving female as well as adult survival was derived directly from the test data.
Significant deviations were determined in comparison to the control using statistical standard procedures as Normality Test, Equal Variance Test and t-test.
Prior to running an analysis of variance test, a normality test and an equal variance test were performed. P-values of the normality tests were 0.001 for reproduction rates and 0.05 for the intrinsic rates of natural increase. P-values for the equal variance tests were 0.05. The a-value (acceptable probability of incorrectly concluding that there is a difference) was a=0.05.
For the determination of significant deviations of the reproduction rates and the intrinsic rates of natural increase t-test (p = 0.05) was used.
The coefficients of variation of alive offspring produced per parent were calculated for the control and the saturated test item solution.

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

                 (n = 10)

Nominal Test Item Concentration [mg/L]	Mortality [%]
	7 days	14 days	21 days
1.00 (Saturated solution)	0	0	10
Control	0	10	10

Number of Juveniles in the Control and Test Group after 21 d

Nominal Test Item Concentration [mg/L]												No. of Parents producing Juveniles	Mean No. of Juveniles per Parent producing Juveniles			Compared versus Control
	Number of Juveniles in Replicate No.										Total
											No.
														CV		Inc.		Stat.
	1	2	3	4	5	6	7	8	9	10	å	N	MV ± SD	[%]		[%]
1.00 (Saturated solution)	121	--	107	132	130	128	122	139	121	130	1130	9	126 ± 9	7		1		no
Control	132	137	130	130	140	--	131	133	98	95	1126	9	125 ± 17		13		—

Relative Number of Dead (Stillborn + Aborted Eggs) to Total Number of Juveniles

Nominal Test Item Concentration [mg/L]	Number of Juveniles			Percentage of Dead Juveniles* [%]
	Dead	Alive	Total
1.00 (Saturated solution)	15	1130	1145	1
Control	2	1126	1128	1

First Appearance of Living Juveniles in the Individual Groups

Nominal Test Item Concentration [mg/L]	Day of First Appearance of Living Juveniles										First Appearance Mean Day
	in Replicate No.
	1	2	3	4	5	6	7	8	9	10
1.00 (Saturated solution)	8	--	8	8	8	8	8	8	8	8	8.0
Control	8	8	8	8	9	--	8	8	8	8	8.1

Total Length and Dry Weight of the Parental Daphnids

Nominal Test Item Concentration [mg/L]	Total Length of the Parental Daphnids [mm]										MV	N	Dry Weight [mg]
	Replicate No.
	1	2	3	4	5	6	7	8	9	10	[mm]		S	MV
1.00 (Saturated solution)	5.25	--	5.25	5.50	5.75	5.50	5.25	5.50	5.50	5.50	5.44	9	8.0	0.89
Control	5.25	5.50	5.25	5.75	5.50	--	5.50	5.50	5.50	5.25	5.44	9	8.6	0.96

Validity criteria fulfilled:

yes

Conclusions:

At the saturated test item solution with 1.00 mg test item/L no biologically or statistically significant effects were determined. The No Observed Effect Concentration (NOEC) and the Lowest Observed Effect Concentration (LOEC) were as follows:
NOEC : 1.00 mg/L
LOEC : > 1.00 mg/L
All provided effect values are based on the nominal concentration of the test item.

Executive summary:

The Daphnia magna Reproduction Test (Semi-Static, 21 d) of the test item was conducted as a limit test with the saturated solution* with 1.00 mg/L test item according to OECD 211 (2008).
The selected test organism was Daphnia magna STRAUS (Clone 5). Ten daphnids individually held were used for the saturated test item solution* and the control, respectively. At test start the daphnids were 2 to 24 h old. The test method was semi-static with a daily renewal of the test solutions. The aim of the Daphnia Reproduction Test over 21 days was to assess effects on the reproduction capacity and other test item-related effects, including occurrence of aborted eggs and stillborn juveniles, time of production of first brood, adult mortality, intrinsic rate of natural increase, dry body weight and length of the parent daphnids.
A specific analytic of the test item was not possible, because the test item is insoluble in water and organic solvents. The results given are based on the nominal concentration of 1.00 mg/L of the test item.

·   The average values of juveniles per parental daphnid were 125 for the control and 126 for the saturated test item solution* with 1.00 mg test item/L after 21 days. The reproductive output of daphnids in the saturated test item solution* (limit concentration) was comparable to the output of daphnids in the control (t-test, p = 0.05).

·   The coefficient of variation of the number of living offspring produced per parent was 13 % for the control and 7 % for the saturated test item solution*.

·   The intrinsic rates of natural increase (IR) of the surviving parent daphnids accounting for generation time and number of offspring were calculated as a measure for population growth and maintenance. The IR of the saturated solution* was comparable to the control group (t-test, p = 0.05).

·   No significant adult mortality (= 20 %) or immobilization of parental daphnids was observed in the control and the saturated solution*. The adult mortality was 10 % in the control and in the saturated test item solution*, respectively.

·   Related to the total number of produced juveniles (dead + alive) the percentage of dead juveniles and aborted eggs was = 1 % at the control and the saturated solution, which is seen to be not biologically significant.

Five broods, respectively, were observed during the test period for all parental daphnids. The first brood was released from all parent daphnids in all treatments until day 9.

·   The mean dry body weight and total body lengths of all parental daphnids of the test and the control group were determined at the end of the study.
The mean dry body weight of the parental daphnids was 0.96 mg per daphnid for the control and 0.89 mg per daphnid for the saturated test item solution.
The mean body length of the parental daphnids was 5.44 mm per daphnid for both, the control and the saturated test item solution.

·   No malesand ephippia (winter eggs) were observed in the respective control or test groups.

A summary of all test item related effects is given in Table 1. No biologically or statistically significant effect could be determined in the saturated test item solution with 1.00 mg/L test item when compared to the control.

Summary of all Test Item Related Effects

                   (Based on the nominal concentration of the test item of 1.00 mg/L)

		Test Item
Effects	Control	Nominal Concentration 1.00 mg/L	Significance
Mean Number of Juveniles per Producing Parent (Reproduction Rate ± SD)	125 ± 17	126 ± 9	no^1)
Coefficient of Variation of the Mean Number of Juveniles per Producing Parent	13	7	no
Intrinsic Rate of Natural Increase (Mean ± SD)	0.48 ± 0.03	0.49 ± 0.02	no^1)
Appearance of First Brood (Mean Day ± SD)	8.1 ± 0.3	8.0 ± 0	—
Number of Broods	5	5	—
Stillborn Juveniles and Aborted Eggs	2	15	no
Adult Mortality after 21 Days [%]	10	10	no
Parent Daphnids: Mean Dry Weight [mg]	0.96	0.89	—
Parent Daphnids: Mean Body Length [mm] ± SD	5.44 ± 0.17	5.44 ± 0.17	—
NOEC	1.00 mg/L

Description of key information

PB25

The Daphnia magna Reproduction Test (Semi-Static, 21 d) of the test item was conducted as a limit test with the saturated solution* with 1.00 mg/L test item according to OECD 211 (2008).
A specific analytic of the test item was not possible, because the test item is insoluble in water and organic solvents. The results given are based on the nominal concentration of 1.00 mg/L of the test item. At the saturated test item solution with 1.00 mg test item/L no biologically or statistically significant effects were determined. The No Observed Effect Concentration (NOEC) and the Lowest Observed Effect Concentration (LOEC) were as follows:

NOEC :            1.00 mg/L

LOEC  :          > 1.00 mg/L