1-butyl-1,2-dihydro-6-hydroxy-4-methyl-2-oxonicotinonitrile

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-03-20 to 2017-04-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Batch number: 12-0014/1
- Content: 97.1 g/100 g (1H-NMR)
- Purity: 99.1% (area-% (HPLC))
- Appearance: fine, greenish to grey powder
- Expiry date: 2022
- Water solubility: 1.10 g/L + 0.05 g/L at T = 20.0°C + 0.5°C

Analytical monitoring:

yes

Details on sampling:

- Sampling method: Sampling at start and end of the test (duplicate samples for chemical analysis and a back-up); 10 mL samples (new media) taken from freshly prepared control and test media flask, at the end of the test, 10 mL samples (old media) taken from pooled control and test vessels
- Sample storage conditions before analysis: frozen until analysis

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: a 120 mg/L test solution (limit concentration) was prepared by adding 60.05 mg of test substance to 500 mL of Elendt M4 medium; stirred for 24 hour at room temperature in the dark (test media code = TM1).
- Controls: negative control (without test item and reference substance), positive control (with reference substance
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): there was slightly more particulate present in the sample for TM1 than in the control. As there was no toxicity observed during the test this difference is not considered to be significant and had no impact on the integrity of the study.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Source: originally obtained from dormant ephippia supplied by MicroBioTests Inc, Belgium
- Container for culturing: cultured in 1 litre glass beakers containing 1000 mL of Elendt M4 medium
- Age of test organisms: less than 24 hours, collected from the second brood onwards
- Health of cultures: highest quality and sensitivity ensured due to tests with a referent toxicant


MAINTENANCE OF CULTURE
- Type of food: concentrated suspension of Chlorella vulgaris
- Feeding frequency: daily during culturing, but not fed during the test
- Culture medium: renewed or partially renewed at least once a week
- Cultures maintained up to a maximum of 42 days (cultures usually discarded at day 35)
- Photoperiod: same as test

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

none

Hardness:

217 mg CaCO3/L

Test temperature:

20.0 - 20.7°C

pH:

6.45 - 7.73

Dissolved oxygen:

79 - 82 mg/L

Conductivity:

724 μS/cm

Nominal and measured concentrations:

- Nominal concentration test item: 120 mg/L (based on range finding information)
- Measured concentration test item: 112 mg/L (t=0 h), 103 mg /L (t=48 h)
- Nominal concentration control: 0 mg/L
- Measured concentration control: value below limit of quantification (t=0 h and t=48 h)

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL glass beakers
- Type: closed, covered with clear plastic petri dish lids
- Material, size, headspace, fill volume: a volume of 50 mL was added to each beaker
- Aeration: none
- No. of organisms per vessel: 5
- Total no. of animals/conc.: 20
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: synthetic freshwater (M4 Medium) on the basis of purified reverse osmosis (RO) water
- Hardness (mg CaCO3/L): 217
- Alkalinity (mg CaCO3/L): 17
- Conductivity (μS/cm): 713
- Dissolved Oxygen (mg/L): 8.66
- pH: 7.62-7.73
- Culture medium different from test medium: no


OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: artificial light, 16-hour light: 8 hour dark cycle with an approximate 30 minute dawn: dusk transition period


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Swimming ability of the test animals, visually after 24 h and 48 h

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 120 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 120 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 108 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 108 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

LOEC

Effect conc.:

> 120 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

> 120 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

LOEC

Effect conc.:

> 108 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

> 108 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

>= 120 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

>= 120 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

>= 108 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

>= 108 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- No effects on mobility were seen at the limit concentration tested
- Other biological observations: no
- Mortality of control: not observed
- Other adverse effects control: no
- Abnormal responses: no
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

Results of control experiment of 12 May 2017:

EC50 (24 h) = 1.37 mg/L
NOEC (24 h) = 1.0 mg/L
LOEC (24 h) = 1.8 mg/L

EC50 (48 h) = 0.626 mg/L
NOEC (48 h) = 0.32 mg/L
LOEC (48 h) = 0.56 mg/L

Reported statistics and error estimates:

No 95% confidence limits have been calculated, as the effect values were greater than the highest concentration.

Table 1: Immobilisation of Daphnia magna after 24 hours exposure during the definitive test

Nominal Concentration (mg/L)	Mean Measured Concentration (mg/L)	Number of Daphnia magna Exposed	Mobile Daphnia magna		Immobile Daphnia magna
			Submerged	Surface	Submerged	Surface
Control	Control	20	20	-	-	-
120	108	20	19	-	1	-

- Not applicable

Table 2: Immobilisation of Daphnia magna after 48 hours exposure during the definitive test

Nominal Concentration (mg/L)	Mean Measured Concentration (mg/L)	Number of Daphnia magna Exposed	Mobile Daphnia magna		Immobile Daphnia magna
			Submerged	Surface	Submerged	Surface
Control	Control	20	19	1	-	-
120	108	20	19	-	1*	-

- Not applicable

* Daphnia magna was immobilised at 24 hours

Validity criteria fulfilled:

yes

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

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

Adequacy of study:

supporting study

Justification for type of information:

Please refer to section "Additional Information" in the Endpoint Summary for "Read Across Justification".

Reason / purpose for cross-reference:

read-across source

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 37.2 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Remarks on result:

other: based on a reported 37.2% active ingridient content in the source substance

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC0

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Reference 3

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2000-12-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)

Qualifier:

according to guideline

Guideline:

ISO 6341 (Water quality - Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea))

GLP compliance:

yes

Specific details on test material used for the study:

CAS number: 28141-13-1

Analytical monitoring:

yes

Details on sampling:

At the start of the test (after 0 h) and at the end of the test (after 48 h) samples from vessels without daphnids were analysed. The concentration control analyses were performed with test solutions of 0 (control), 12.5 and 100 mg/L test substance.

Vehicle:

no

Details on test solutions:

The test substance was stirred in M4 medium for about 20 hours at 20±2 °C. Undissolved test substance was removed by centrifugation (approximately 20 min at about 17700 G). The nominal concentration of the eluate was 100 mg/L. By diluting this eluate with M4 medium the nominal test concentrations (mg/L) were prepared.

Test organisms (species):

Daphnia magna

Details on test organisms:

- Source: The clone of Daphnia magna STRAUS (1820) used was supplied by the lnstitut National de Recherche Chimique Appliqué France, in 1978. From this date on this clone was cultured and bred continuously in the Laboratory Experimental Toxicology and Ecology of BASF AG in Ludwigshafen.
- Age of the animals at the start of the test: 2 - 24 h (starting with the 3rd breed of parent animals)
- Age of stock animals: 2-4 weeks

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

2.44 mmol/L (Total hardness of M4 medium used)

Test temperature:

20.7 - 21.7 °C

pH:

7.8 - 8.0

Dissolved oxygen:

8.7 - 8.8 mg/L

Nominal and measured concentrations:

- Nominal concentrations: 0 (control), 12.5, 25, 50 and 100 mg/L (based on range-finding test). Analytical verification was performed with test solutions of 0 (control), 12.5 and 100 mg/L.
- Measured concentrations (t=0h): <1, 13.2 and 102.0 mg/L
- Measured concentrations (t=48h): <1, 13.3 and 101.9 mg/L

Details on test conditions:

TEST DESIGN:
- Test vessel: Test tubes (glass) with flat bottom (nominal volume 20 mL)
- Test volume: 10 mL
- Total number of animlas/conc.: 20
- Number of animals/vessel: 5
- Loading (animals/mL): 0.5
- Number of replicates/conc.: 4
- Number of replicates/control: 4
- Feeding: not during the test

TEST MEDIUM / WATER PARAMETERS:
Synthetic freshwater was used for culture and test purposes. This water was prepared on the basis of an ultrapure, demineralized water with a conductivity <0.05 µS/cm. This M4 medium was prepared according to guideline and has the following properties: Total hardness: 2.20-3.20 mmol/L; Alkalinity up to pH 4.3: 0.80-1.00 mmol/L; Molar ratio Ca/Mg: about 4:1; pH value: 7.5-8.5; Conductivity: 550-650 µS/cm. After preparation, the M4 medium was aerated for approximately 24 h until saturation with oxygen was reached.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Illumination: artificial light, type warm white (e.g. OSRAM L58 W31) day:night rhythm = 16 : 8 h
- Light intensity: about 2 - 7 µE/(m²·s) at a wave length of 400 - 700 nm

EFFECT PARAMETERS MEASURED:
- Swimming ability of the test animals, visually after 0, 24 and 48 h

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 37.2 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Remarks on result:

other: based on a reported 37.2% active ingredient content in the test substance

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC0

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

No effects on mobility were seen at any of the concentrations tested.

Results with reference substance (positive control):

24-h EC50 = 1.32 mg/L (control experiment of 13 July 2000)

Validity criteria fulfilled:

yes

Description of key information

The 48-h EC50 value of the test substance is >120 mg/L (nominal) in aquatic invertebrates (D. magna).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

120 mg/L

Additional information

The acute immobilisation of the test substance to Daphnia magna (STRAUS) was determined under GLP conditions according to OECD Guideline 202 (BASF SE, 2017). The test was performed in a static and closed system as a limit-test over a duration of 48 hours with the only nominal test concentration of 120 mg/L (measured mean concentration: 108 mg/L), along with a laboratory negative control (0 mg/L). Four replicates with five daphnids each for the test concentration and the negative controls were used. Mobility of daphnids was recorded after 24 and 48 hours of exposure. Based on the nominal concentrations of the test substance, the 48-h EC50 was determined to be >120 mg/L. All validity criteria were fulfilled.

In addition, to assess the ecotoxicological potential of the test substance, read-across data available for the close structural analogue 1-Ethyl-2-hydroxy-3-cyan-4-methylpyridon-6 (CAS 28141-13-1) were considered. This analogue differs from the substance only in that it has an ethyl- instead of a butyl-chain attached to the nitrogen atom in the pyridine ring. The acute toxicity to aquatic invertebrates was determined in a study according to OECD guideline No 202 and in compliance with GLP criteria (BASF AG, 1999). In this study daphnids (D. magna, 20 per concentration) were exposed to nominal test substance concentrations of 0 (control), 12.5, 25, 50 and 100 mg/L for 48 hours under static conditions. Analytical confirmation of the nominal test concentrations of 12.5 and 100 mg/L at the start and the end of the test showed that test concentrations remained well within ±20% of nominal concentrations. Therefore, effect concentrations are expressed as nominal. Mobility of daphnids was recorded after 24 and 48 hours exposure. No immobilization of daphnids was observed in any of the concentrations tested. Based on these findings the 48-h EC50 value is >100 mg/L (nominal). Based on a test substance purity of 37.2%, this value is recalculated at >37.2 mg/L for the active ingredient. As the structural analogue differs from the target substance only in that it has an ethyl- instead of a butyl-chain attached to the nitrogen atom in the pyridine ring, it may be considered likely that this 48-h EC50 value applies also to the test substance 1-butyl-1,2-dihydro-6-hydroxy-4-methyl-2-oxonicotinonitrile.

Based on the results of the Acute Immobilisation Test with Daphnia magna (BASF SE, 2017), the test substance is considered to be not toxic to aquatic invertebrates since no aquatic toxicity was observed at 120 mg/L. This result is supported by the results for the structural analogue as no effects were observed up to the highest concentration tested.