N-(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)-2-[(2-methoxyphenyl)azo]-3-oxobutyramide

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-10-14 to 2011-10-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed according to relevant guidelines and compliant to GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

according to German Chemikaliengesetzt and directive 88/320/EEC

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The test item was analytically verified from the limit concentration and the control from freshly prepared media after 0 and 72 h and from 24 h old media after 24 and 96 h.
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 C° until analysis, if necessary.

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. Weighing of the test item for day 0 and 1 was carried out 72 h prior to test start and stored in the dark at room temperature until preparation of the saturated solution. Weighing of the test item for day 2 and 3 was carried out after 0 and 24 h of the study, respectively.
The stock solution was stirred for 48 hours with a magnetic stirrer. The undissolved particles were removed by membrane filtration (membrane filter 0.45 µm, RC, MACHEREY-NAGEL). On day 0, centrifugation for 20 min at 10000 g was performed prior to filtration. No pellet was generated, so the procedure was declared insufficient. On the following days only membrane filtration was used
- Eluate: Dilution water
- Differential loading: None, limit test
- Controls: 7 fish in dilution water (without test item) were tested under the same test conditions as the test replicates.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Source: All fish used in the test were gained at DR.NOACK-LABORATORIEN from a single brood stock. (supplier: Niedersächsischer Landesbetrieb für Wasserwirtschaft, Küsten- und Naturschutz, An der Scharlake 39, D-31135 Hildesheim)
- Length at study initiation (length definition, mean, range and SD): Average body length: 2.19 cm
- Weight at study initiation (mean and range, SD): Average body weight: 0.123 g
- Method of breeding: Holding was performed at the test facility at 23 +/- 2 °C and diffuse light (0.1 - 10 µmol photons • m-2 • s-1, natural photoperiod). The water was changed at least once per week. The dissolved oxygen concentration was more than 80 % of the air saturation value. No disease treatments were administered throughout holding and testing. Food was provided 3 times per week. The amount of food was 4 % of the fish body weight per feeding day. The test fish were not fed 24 h before the test started.
- Feeding during test: No feeding during test



ACCLIMATION
- Acclimation period: Zebrafish with at least 12 days of acclimatisation and mortality < 5 % within these days before the study starts were used in the test.
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: 4 % of the fish body weight per feeding day. The test fish were not fed 24 h before the test started. Food: Sera Vipan; SERA GMBH, D-52518 Heinsberg
- Feeding frequency: 3 times per week.
- Health during acclimation (any mortality observed): No mortality observed, no disease treatments were administered throughout holding and testing.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

Total Hardness at day 0: 52 mg/L

Test temperature:

Please refer to "Any other information on materials and methods"

pH:

Please refer to "Any other information on materials and methods"

Dissolved oxygen:

Please refer to "Any other information on materials and methods"

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: Glass aquaria
- Type (delete if not applicable): open, loosely covered by glass tops
- Material, size, headspace, fill volume: glass, 2 L test volume per vessel
- Aeration: Gentle aeration
- Renewal rate of test solution (frequency/flow rate): daily renewal (after 0, 24, 48, 72 hours)
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: less than 1 g fish per L test solution


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap water of local origin was used for holding and testing. The water was filtered on activated charcoal and aerated for at least 24 h to remove possible chlorine.
Nominal water parameters:
Total hardness: 10 - 250 mg CaCO3/L
pH-value: 6.0 - 8.5
- Culture medium different from test medium: no
- Intervals of water quality measurement: pH-value, temperature and oxygen saturation were measured in all vessels at the beginning of the test and every 24 h (from old and new media). Total hardness of the water was determined at the beginning out of the test control. During the test the water temperature was recorded continuously with a data logger (once per hour).


OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: natural
- Light intensity: 0.1 - 10 µmol photons x m-2 x s-1


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Fish were considered dead if there was no visible movement (e.g. gill covers movement) and if touching of the caudal peduncle produced no reaction. Records were kept of visible abnormalities (e.g. loss of equilibrium, swimming behaviour, respiratory function, pigmentation, etc.). Observations were made after 24, 48, 72 and 96 h.


TEST CONCENTRATIONS
- Range finding study:
Cumulative Mortality [%] in the Preliminary Test (non GLP)(n = 5)

Nominal
concentration level
[mg/L] Test Duration [hours]
24 48 72 96
1.00 0 0 0 0
Control 0 0 0 0

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

EC0

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mortality, appearance and behaviour

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: Not observed
- Mortality of control: 0%
- Other adverse effects control: Not observed
- Abnormal responses: Not observed
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The test item solutions were clear.

Results with reference substance (positive control):

No reference substance tested

Reported statistics and error estimates:

The concentration levels leading to 0 and 100 % mortality (LC0 and LC100) after 96 h were determined directly from the test results. LC50-values have not to be calculated in a limit test.

Sublethal observations / clinical signs:

Observations in the Test Vessels

                        (n=7)

Nominal concentration level [mg/L]	Effect*)	Test Duration [h]
		24	48	72	96
1.00	(1)	7/7	7/7	7/7	7/7
Control	(1)	7/7	7/7	7/7	7/7

*) the number in brackets correspond to the ffollowing observation: (1) = normal behaviour

Cumulative Mortality [%] in the Test Vessels

           

Nominal concentration level [mg/L]	Test Duration [h]
	24	48	72	96
1.00	0	0	0	0
Control	0	0	0	0

LC-Values with 95 % Confidence Intervals

                        based on nominal concentration

Test duration [h]	LC50		p = 95 %
24	> 1.00 mg/L		not applicable
48	> 1.00 mg/L		not applicable
72	> 1.00 mg/L		not applicable
96	> 1.00 mg/L		not applicable
LC100 =		> 1.00 mg/L
Lowest tested concentration level
with 100 % mortality after 96 h
LC0 =		1.00 mg/L
Highest tested concentration level
with 0 % mortality after 96 h

Validity criteria fulfilled:

yes

Conclusions:

In this study the test item did not cause any effects to zebrafish after 96 h when testing a saturated solution with a nominal concentration level of 1.00 mg/L of the test item. The LC0 (0 – 96 h) was 1.00 mg/L. The very poor solubility of the test item precluded the analytical determination of the test item in the saturated solution (below limit of quantification).

Executive summary:

The acute toxicity of the test item to fish (zebrafish) was determined according to OECD-Guideline for Testing of Chemicals No. 203.

A semi-static limit test with daily renewal of the test media was conducted with a saturated solution* of 1.00 mg/L of the test item. Duration of the test was 96 h. 7 test organisms were exposed to the test concentration level and the control. Water quality parameters pH-value, temperature and oxygen-saturation measured after 0, 24, 48, 72 and 96 h were determined to be within the acceptable limits.

The concentration of the test item was determined via HPLC - DAD from freshly prepared media after 0 and 72 h and from the corresponding 24 h old media after 24 and 96 h. The acute toxicity of the test item is summarised in the following table:

LC-Values with 95 % Confidence Intervals

                                based on nominal concentration

Test duration [h]	LC50		p = 95 %
24	> 1.00 mg/L		not applicable
48	> 1.00 mg/L		not applicable
72	> 1.00 mg/L		not applicable
96	> 1.00 mg/L		not applicable
LC100 =		> 1.00 mg/L
Lowest tested concentration level
with 100 % mortality after 96 h
LC0 =		1.00 mg/L
Highest tested concentration level
with 0 % mortality after 96 h

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-10-17 to 2011-10-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed according to relevant guidelines and compliant to GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

and Council Regulation (EC) No. 440/2008 Method C.1

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

according to German Chemikaliengesetzt and directive 88/320/EEC

Analytical monitoring:

yes

Details on sampling:

- Sampling method: The test item concentration was analytically verified from the limit concentration and the control from freshly prepared media after 0 and 72 h and from 24 h old media after 24 and 96 h.
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 C° until sample preparation, if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution (1 mg/L test item was weighed out) was prepared with dilution water. The stock solution was stirred for 48 h with a magnetic stirrer. The undissolved particles were removed by membrane filtration (membrane filter 0.45 µm, RC Macherey-Nagel). The centrifugation for 20 min at 10000 g was performed at day 0. No pellet was generated, so the procedure was declared insufficient. On the following days only membrane filtration was used.
- Eluate: Dilution water
- Differential loading: Saturated solution of 1 mg/L
- Controls: 7 fish in dilution water (without test item) were tested under the same test conditions as the test replicates.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Source: All fish used in the test were gained at DR.NOACK-LABORATORIEN from a single brood stock. (supplier: Niedersächsischer Landesbetrieb für Wasserwirtschaft, Küsten- und Naturschutz, An der Scharlake 39, D-31135 Hildesheim)
- Length at study initiation (length definition, mean, range and SD): Average body length: 2.19 cm
- Weight at study initiation (mean and range, SD): Average body weight: 0.123 g
- Method of breeding: Holding was performed at the test facility at 23+/-2 °C and diffuse light (0.1 - 10 µmol photons • m-2 • s-1, natural photoperiod). The water was changed at least once per week. The dissolved oxygen concentration was more than 80 % of the air saturation value. No disease treatments were administered throughout holding and testing. Food was provided 3 times per week. The amount of food was 4 % of the fish body weight per feeding day. The test fish were not fed 24 h before the test started.
- Feeding during test: No feeding during test



ACCLIMATION
- Acclimation period: Zebrafish with at least 12 days of acclimatisation and mortality < 5 % within these days before the study starts were used in the test.
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: 4 % of the fish body weight per feeding day. The test fish were not fed 24 h before the test started. Food: Sera Vipan; SERA GMBH, D-52518 Heinsberg
- Feeding frequency: 3 times per week.
- Health during acclimation (any mortality observed): No mortality observed, no disease treatments were administered throughout holding and testing.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

Total Hardness at day 0: 51.6 mg/L

Test temperature:

Please refer to "Any other information on materials and methods"

pH:

Please refer to "Any other information on materials and methods"

Dissolved oxygen:

Please refer to "Any other information on materials and methods"

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Please refer to "Any other information on materials and methods"

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass aquaria
- Type (delete if not applicable): open, loosely covered by glass tops
- Material, size, headspace, fill volume: glass, 2 L test volume per vessel
- Aeration: Gentle aeration
- Renewal rate of test solution (frequency/flow rate): daily renewal (after 0, 24, 48, 72 hours)
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: less than 1 g fish per L test solution


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap water of local origin was used for holding and testing. The water was filtered on activated charcoal and aerated for at least 24 h to remove possible chlorine.
Nominal water parameters:
Total hardness: 10 - 250 mg CaCO3/L
pH-value: 6.0 - 8.5
- Culture medium different from test medium: no
- Intervals of water quality measurement: pH-value, temperature and oxygen saturation were measured
measurements in all vessels at the beginning of the test and every 24 h (from old and new media). Total hardness of the water was determined at the beginning out of the test control. During the test the water temperature was recorded continuously with a data logger (once per hour).


OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: natural
- Light intensity: 0.1 - 10 µmol photons x m-2 x s-1


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Fish were considered dead if there was no visible movement (e.g. gill covers movement) and if touching of the caudal peduncle produced no reaction. Records were kept of visible abnormalities (e.g. loss of equilibrium, swimming behaviour, respiratory function, pigmentation, etc.). Observations were made after 24, 48, 72 and 96 h.


TEST CONCENTRATIONS
- Range finding study: Yes
Cumulative Mortality [%] in the Preliminary Test (non GLP)
(n = 5)

Nominal loading level
[mg/L] Test Duration [hours]
24 48 72
1.00 0 0 0
Controls 0 0 0

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

EC0

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mortality and sublethal effects

Remarks on result:

other: Due to the very poor solubility of the test item, quantitative analytical determination was precluded (below LOQ)

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: Due to the very poor solubility of the test item, quantitative analytical determination was precluded (below LOQ)

Details on results:

- Behavioural abnormalities: Not observed
- Mortality of control: 0%
- Other adverse effects control: Not observed
- Abnormal responses: Not observed
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The test item was clearly dissolved.

Results with reference substance (positive control):

No reference substance tested

Reported statistics and error estimates:

The concentration leading to 0 and 100 % mortality (LC0 and LC100) after 96 h were determined directly from the test results. LC50-values have not to be calculated in a limit test.

Sublethal observations / clinical signs:

Observations in the Test Vessels

                        (n=7)

Nominal loading [mg/L]	Effect*)	Test Duration [h]
		24	48	72	96
1.00	(1)	7/7	7/7	7/7	7/7
Control	(1)	7/7	7/7	7/7	7/7

*) The number in brackets correspond to the following observation: (1) = Normal behaviour

Cumulative Mortality [%] in the Test Vessels

           

Nominal loading [mg/L]	Test Duration [h]
	24	48	72	96
1.00	0	0	0	0
Control	0	0	0	0

LC-Values with 95 % Confidence Intervals

                        (nominal loading)

Test duration [h]	LL50		p = 95 %
24	> 1.00 mg/L		not applicable
48	> 1.00 mg/L		not applicable
72	> 1.00 mg/L		not applicable
96	> 1.00 mg/L		not applicable
LC100 =		> 1.00 mg/L
Lowest test item concentration
with 100 % mortality after 96 h
LC0 =		1.00 mg/L
Highest test item concentration
with 0 % mortality after 96 h

Validity criteria fulfilled:

yes

Conclusions:

In this study the test item did not cause any effects to Zebrafish after 96 h when tested as a saturated solution with a nominal loading level of 1.00 mg/L. The LC0 (0 – 96 h) was 1.00 mg/L

Executive summary:

The acute toxicity of the test item to fish (Zebrafish) was determined according to OECD-Guideline for Testing of Chemicals No. 203 (1992).

A semi-static limit test with daily renewal of the test media was conducted with a saturated solution * of 1.00 mg/L of the test item. Duration of the test was 96 h. 7 test organisms were exposed to the test loading level and the control. Water quality parameters pH-value, temperature and oxygen-saturation measured after 0, 24, 48, 72 and 96 h were determined to be within the acceptable limits.

The concentration of the test item was determined via HPLC - DAD from freshly prepared media after 0 and 72 h and from the corresponding 24 h old media after 24 and 96 h. The acute toxicity of the test item is summarised in the following table:

             Table 1:        LC-Values with 95 % Confidence Intervals

                                   (nominal loading)

Test duration [h]	LC50		p = 95 %
24	> 1.00 mg/L		not applicable
48	> 1.00 mg/L		not applicable
72	> 1.00 mg/L		not applicable
96	> 1.00 mg/L		not applicable
LC100 =		> 1.00 mg/L
Lowest test item concentration
with 100 % mortality after 96 h
LC0 =		1.00 mg/L
Highest test item concentration
with 0 % mortality after 96 h

Reference 3

Endpoint:

short-term toxicity to fish

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

see Rationale and Justification for the Analogue Read-Across Approach used for the Registration of Acetolone Pigments – Nanoforms and Bulk Forms (Chapter 13)

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Key result

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

<= 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: Read across from bulk PO36

Validity criteria fulfilled:

yes

Conclusions:

PY120 (Nanoform)
In this study the test item did not cause any effects to zebrafish after 96 h when testing a saturated solution with a nominal concentration level of 1.00 mg/L of the test item. The LC0 (0 – 96 h) was 1.00 mg/L. The very poor solubility of the test item precluded the analytical determination of the test item in the saturated solution (below limit of quantification).
PO36 (Bulk)
In this study the test item did not cause any effects to Zebrafish after 96 h when tested as a saturated solution with a nominal loading level of 1.00 mg/L. The LC0 (0 – 96 h) was 1.00 mg/L

Reference 4

Endpoint:

short-term toxicity to fish

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

see Rationale and Justification for the Analogue Read-Across Approach used for the Registration of Acetolone Pigments – Nanoforms and Bulk Forms (Chapter 13)

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Key result

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

<= 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: Read across from bulk PO36

Validity criteria fulfilled:

yes

Conclusions:

PY120 (Nanoform)
In this study the test item did not cause any effects to zebrafish after 96 h when testing a saturated solution with a nominal concentration level of 1.00 mg/L of the test item. The LC0 (0 – 96 h) was 1.00 mg/L. The very poor solubility of the test item precluded the analytical determination of the test item in the saturated solution (below limit of quantification).
PO36 (Bulk)
In this study the test item did not cause any effects to Zebrafish after 96 h when tested as a saturated solution with a nominal loading level of 1.00 mg/L. The LC0 (0 – 96 h) was 1.00 mg/L

Description of key information

Fish

PY120 (Nanoform)

In this study the test item did not cause any effects to zebrafish after 96 h when testing a saturated solution with a nominal concentration level of 1.00 mg/L of the test item. The LC0 (0 – 96 h) was 1.00 mg/L. The very poor solubility of the test item precluded the analytical determination of the test item in the saturated solution (below limit of quantification).

PO36 (Bulk)

In this study the test item did not cause any effects to Zebrafish after 96 h when tested as a saturated solution with a nominal loading level of 1.00 mg/L. The LC0 (0 – 96 h) was 1.00 mg/L