5-(3-Phenylpropioloyl)-1,3-isobenzofurandione

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 July 2016 to 02 February 2017

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)

Version / remarks:

OECD Guideline for Testing of Chemicals, Daphnia sp., Acute Immobilisation Test, reference 202, OECD 2004.

Deviations:

yes

Remarks:

See "any other information" for details

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Lot/Batch Number: NEX-X61-A06 (First batch)*; NEX-X61-A12 (Second batch)**
Expiry Date: 2016-01-09 (First batch)*; 29th April 2017 (Second batch)**
*First batch was used in all range-finding tests carried out.
** Second batch used in definitive test.

Analytical monitoring:

yes

Details on sampling:

Duplicate samples were taken at the start and end of the 48 hour exposure period. Samples were taken from remaining test media after filling test vessels at 0 hours and pooled replicate vessels for 48 hours.

Vehicle:

no

Details on test solutions:

Information provided by the Sponsor stated the sample was insoluble in water.
Two initial range-finding tests were carried out. The first was conducted from 13 to 15 July 2016 but had to be repeated as the time period for stirring the stock solution was insufficient. The second range-finding test was carried out from 27 to 29 July 2016 . In this test a water-accommodated fraction (WAF) of 100mg/l was prepared by adding 0.0501g of the test substance to Elendt M4 medium in an aspirator. This was stirred for 48 hours and was then left to settle for 4 hours and 15 minutes. After settling the aqueous phase was drawn off for testing leaving any sedimented/undissolved material behind. The solution was filtered using a Whatman 54 filter, with the initial 100ml of filtrate discarded. The lower doses (0.1, 1.0 and 10mg/l) were then prepared by dilution of the 100mg/l aqueous phase with Elendt M4 medium, this was considered to be the most practical method for the low concentrations required.
Since an effect was seen at 100mg/l (one Daphnia immobilised and one trapped at the surface) in the second range-finding test, a third range-finding test at concentrations of 0 (Control), 10, 32, 100, 320 and 1000mg/l was carried out from 01 to 03 September 2016. A water-accommodated fraction (WAF) of 1000mg/l was prepared by adding 0.5000g of the test substance to Elendt M4 medium in an aspirator. This was stirred for 48 hours and was then left to settle for 4 hours. After settling the aqueous phase was drawn off for testing leaving any sedimented/undissolved material behind. Since a lot of suspended material was present, approximately five Whatman 54 filters were need to remove the suspended material, with the initial 150ml of filtrate discarded. The test concentrations of 10, 32, 100 and 320mg/l were then prepared by dilution of the 1000mg/l aqueous phase with Elendt M4 medium.
For the definitive test a water-accommodated fraction (WAF) of 1000mg/l was prepared by adding 1.0002g of the test substance to Elendt M4 medium in an aspirator. This was stirred for 69 hours and 5 minutes and was then left to settle for 26 hours (in the range-finding test most of the test substance remained in suspension after a settling period of four hours, a longer settling period of at least 24 hours was therefore proposed in the definitive test). After settling the aqueous phase was drawn off for testing leaving any sedimented/undissolved material behind. The solution was filtered using a Whatman 54 filter, with the initial 100ml of filtrate discarded. The test concentrations of 10, 32, 100 and 320mg/l were then prepared by dilution of the 1000mg/l aqueous phase with Elendt M4 medium.

Test organisms (species):

Daphnia magna

Details on test organisms:

Test organism
Test species: Daphnia magna
Source: From cultures maintained at Chemex Environmental International Ltd since July 2014 and originally sourced from a clone of genotype 5.
Date of gravid isolation: 30 January 2017
Age of Daphnia at start of test: ≤ 24 hours.
Culture conditions: Cultured under static conditions
Temperature: 19.7 – 21.6°C
Dissolved oxygen:>60% ASV
Photoperiod: 16 hours light and 8 hours dark
Feeding regime: Each working day, a suspension of Chlorella vulgaris. The Daphnia were not fed during the test.

Test and culture medium: The Daphnia stocks were cultured, and the tests performed, in Elendt M4 medium as described in OECD Test Guideline 202.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

Not specified

Test temperature:

Temperature range within incubator throughout test: 19.9-20.9°C
(Required: 20±2°C maintained to ±1°C)

pH:

Initial pH of Elendt M4 Daphnia medium: 8.55
(Required: 6-9)
Range in test: 3.85-7.54

Dissolved oxygen:

Dissolved Oxygen in control at 0 Hours: 6.90mg/l
Range in test: 6.08-8.68mg/l
(Required: ≥3mg/l)

Salinity:

Not required - freshwater study

Conductivity:

Not specified

Nominal and measured concentrations:

Nominal: 0 (control), 10, 32, 100, 320, and 1000mg/l

Details on test conditions:

The test was carried out according to OECD Test Guideline 202 (1) and the procedures given below.
Chemex SOP reference: E201 Daphnia magna 24 & 48 hr EC50 acute immobilisation test.
Preliminary test method: Three preliminary (range-finding) tests were carried out in total. The first took place between 13 and 15 July 2016 and due to a small-procedural error had to be repeated. The second range-finding test was carried out from 27 to 29 July 2016 at concentrations of 0 (Control), 0.1, 1, 10 and 100mg/l.
The duration of the test was 48±1 hours with a single replicate at each concentration. The results showed that one animal was immobilised and one was trapped at the surface at 100mg/l. The single animal immobilised at 100mg/l could have been due to a random effect. Therefore, to check this a third range-finding test was conducted from 01 to 03 September 2016 at concentrations of 0 (Control), 10, 32, 100, 320 and 1000mg/l. The duration of the range-finding test was 48±1 hours with two replicates per concentration.
Preliminary test results: Data from the preliminary test identified the 48 hour EL50 as being between 100 and 320mg/l.

Definitive test method:
Test period: 31 January to 02 February 2017
Test duration: 48 ± 1 hour
Test volume: 25ml
Test vessel: 40ml glass vessels. To limit aerial contamination of the test vessels and reduce evaporative losses, the vessels were covered with a perspex sheet.
Number of replicates: Four replicates at each concentration.
Test concentrations: 0 (control), 10, 32, 100, 320, and 1000mg/l
Test organism: Five Daphnia were transferred to each control and test concentration vessel using a pasteur pipette or glass tube in a small amount of culture water (<0.5ml).
Test medium: Elendt M4 Daphnia medium
Replacement regime: Static
Test conditions: Illumination: 16 Hours Light, 8 Hours Dark Cycle
Water quality measurements: The pH (to 0.01), dissolved oxygen mg/l (to 0.01) and temperature (to 0.1°C) measurements were taken at 0 hours on remaining test solution, and at 48-hour endpoint on pooled replicates for each test and control solution. Temperature of incubation was recorded at 0, 24 and 48 hours.

Observation frequency: The number of immobilised Daphnia was recorded after 24 and 48 hour exposure periods.

Calculation of results: Where possible, the EL(r)10, EL(r)50 EL(r)90 values with 95% confidence limits and NOEC(r) were calculated according to the statistical methods of ToxCalc™ Version 5.0 “Comprehensive Toxicity Data Analysis and Database Software”, copyright 1994-1996.

Reference Substance: A separate GLP study (ENV 11395) run between 18 to 20 January 2017 was performed to monitor the sensitivity of the test animals and test procedures.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

354.94 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: All concentrations are expressed as nominal loading rates from water accommodated fractions (WAFs)

Key result

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: All concentrations are expressed as nominal loading rates from water accommodated fractions (WAFs)

Details on results:

The 24-hour EL50 and 48-hour EL50 of Phenylacetylene modified trimellitic anhydride (PETA) including the hydrolysis product to Daphnia magna were 561.62mg/l and 354.94mg/l (both determined by Trimmed Spearman-Karber) respectively. Graphical representations of the 0-48 hour EL50 and 0-48 hour dose-response plot are given in Graphs 1 and 2, respectively.
The 0 to 24-hour NOELR and LOELR were 320mg/l and 1000mg/l respectively (determined by Steel’s Many-One Rank Test).
The 0 to 48-hour NOELR and LOELR were 100mg/l and 320mg/l respectively (determined by Steel’s Many-One Rank Test).
The Daphnia were examined at 24 hours and 48 hours for abnormal behaviour during the determination of immobility. Abnormalities were observed at 24-hours at 320mg/l where all five Daphnia in replicate 3 were lighter in colour and moving slower. Abnormalities were also seen after 48-hours, including two Daphnia in the control recorded as being mobile but trapped in the meniscus and two Daphnia appearing to be moving slower at 320mg/l.
The lowest test concentration which immobilised all twenty Daphnia after 48 hours was 1000mg/l.
The highest concentration where no immobilisation occurred was 100mg/l.
Whilst two (10%) of the twenty control Daphnia were observed as being trapped in the meniscus at 48-hours, these remained mobile. Therefore, none (0%) of the twenty control Daphnia maintained in dilution water were immobilised. This fulfills the control validity criteria of the study as the percentage of immobilisation did not exceed 10%, confirming that the test organisms were of suitable “health” for use in the study.

Results with reference substance (positive control):

The reference study gave a 24-hour EC50 of 1.4mg/l of potassium dichromate which was within the acceptable range 0.6 mg/l to 2.1 mg/l as specified in Technical Corrigendum to ISO6341.

Reported statistics and error estimates:

Where possible, the EL(r)10, EL(r)50 EL(r)90 values with 95% confidence limits and NOEC(r) were calculated according to the statistical methods of ToxCalc™ Version 5.0 “Comprehensive Toxicity Data Analysis and Database Software”, copyright 1994-1996.

 Cumulative immobilisation

Nominal Concentration mg/l	Number immobilised		% immobilisation
	24 hours	48 hours	24 hours	48 hours
0 (Control)	0	0	0	0
10	1	1	5	5
32	0	0	0	0
100	0	0	0	0
320	0	8	0	40
1000	20	20	100	100

 

Observations

Nominal Concentration mg/l	Recorded observations
	24 hours	48 hours
0 (Control)	20NS	18NS2TM
10	19NS	19NS
32	20NS	20NS
100	20NS	20NS
320	15NS, 5LIand Ms	10NS2M^s
1000	-	-

 

Key to comments
S	Sedimentation	Ms	Movement Slower
D	Debris attached to Daphnia	TM	Trapped in Meniscus (mobile)
L^I	Lighter Colouration	TI	Trapped in Meniscus (immobile)
NS	No Signed of Abnormality	SM	Daphnia Smaller
-	All Daphnia immobilised

 

Water quality data

Concentration (mg/l)	0 hours			48 hours
	pH	DO (mg/l)	Temp (°C)	pH	DO (mg/l)	Temp (°C)
0 (Control)	7.23	6.90	20.9	7.11	6.08	21.2
10	7.38	7.86	21.0	7.32	6.60	21.4
32	7.43	8.67	21.3	7.40	6.55	21.5
100	7.41	8.68	21.3	7.51	6.39	21.6
320	7.04	7.28	21.3	7.54	6.69	21.6
1000	3.85	7.91	20.5	4.30	7.40	21.6

 

Within incubator	Temperature (°C)
	0 hours	24 hours	48 hours
Current	20.9	20.3	20.3
Minimum		20.2	20.2
Maximum		20.3	20.3
Within incubator	Light intensity (Lux)
	518		850

 

Note: Water quality was determined on pooled replicates for the test and control solution at the end of the 48 hour test period and on excess solution at the start of the study.

Validity criteria fulfilled:

yes

Conclusions:

The definitive test conducted from 31 January to 02 February 2017 was performed according to the OECD 202 2004 guideline and met all validity criteria.
The 24-hour EL50 and 48-hour EL50 of Phenylacetylene modified trimellitic anhydride (PETA) including the hydrolysis product to Daphnia magna were 561.62mg/l and 354.94mg/l (both determined by Trimmed Spearman-Karber) respectively. Graphical representations of the 0-48 hour EL50 and 0-48 hour dose-response plot are given in Graphs 1 and 2, respectively.
The 0 to 24-hour NOELR and LOELR were 320mg/l and 1000mg/l respectively (determined by Steel’s Many-One Rank Test).
The 0 to 48-hour NOELR and LOELR were 100mg/l and 320mg/l respectively (determined by Steel’s Many-One Rank Test).
The Daphnia were examined at 24 hours and 48 hours for abnormal behaviour during the determination of immobility. Abnormalities were observed at 24-hours at 320mg/l where all five Daphnia in replicate 3 were lighter in colour and moving slower. Abnormalities were also seen after 48-hours, including two Daphnia in the control recorded as being mobile but trapped in the meniscus and two Daphnia appearing to be moving slower at 320mg/l.
The lowest test concentration which immobilised all twenty Daphnia after 48 hours was 1000mg/l.
The highest concentration where no immobilisation occurred was 100mg/l.
Whilst two (10%) of the twenty control Daphnia were observed as being trapped in the meniscus at 48-hours, these remained mobile. Therefore, none (0%) of the twenty control Daphnia maintained in dilution water were immobilised. This fulfills the control validity criteria of the study as the percentage of immobilisation did not exceed 10%, confirming that the test organisms were of suitable “health” for use in the study.

Executive summary:

This section summarises aquatic toxicity test results obtained by Chemex Environmental International Limited on a sample as detailed below:

Substance under test: Phenylacetylene modified trimellitic anhydride (PETA) including the hydrolysis product

Test species: Daphnia magna

Test type: Acute toxicity: 48 hour EL50.

Protocol: OECD 202: Daphnia sp. Acute Immobilisation Test

Experimental period:

Range-finding tests: 1) 11 to 15 July 2016

2) 25 to 29 July 2016

3) 30 August to 03 September 2016

Definitive test: 27 January to 02 February 2017

Test concentrations: 0 (control), 10, 32, 100, 320, and 1000mg/l

Results: All study validity criteria were met. The results are summarised in the table below.

 

Period of exposure	EL50value (with 95% confidence limits)
24 hour	561.62 mg/l (545.55-578.15 mg/l)
48 hour	354.94 mg/l (272.83-461.75 mg/l)
48 hour NOELR	100 mg/l (Steel’s Many-One Rank Test)*
48 hour 100% immobilisation	1000 mg/l

Methods used in ToxCal v5.0: Both 24-hour and 48-hour EL50’s determined by Trimmed Spearman-Karber.

*Following Shapiro-Wilk’s Test which indicated non-normal distribution. Equality of variance could not be confirmed.

All concentrations are expressed as nominal loading rates from water accommodated fractions (WAFs).

Description of key information

The substance is known to hydrolyse relatively rapidly in water, hence both the substance and the associated hydrolysis product were assessed within the test.

The 48-hour EL50 of Phenylacetylene modified trimellitic anhydride (PETA) including the hydrolysis product to Daphnia magna was 354.94mg/l.

The 0 to 48-hour NOELR and LOELR were 100mg/l and 320mg/l respectively.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

354.94 mg/L

Additional information

This section summarises aquatic toxicity test results obtained by Chemex Environmental International Limited on a sample as detailed below:

Substance under test: Phenylacetylene modified trimellitic anhydride (PETA) including the hydrolysis product

Test species: Daphnia magna

Test type: Acute toxicity: 48 hour EL50.

Protocol: OECD 202: Daphnia sp. Acute Immobilisation Test

Experimental period:

Range-finding tests: 1) 11 to 15 July 2016

2) 25 to 29 July 2016

3) 30 August to 03 September 2016

Definitive test: 27 January to 02 February 2017

Test concentrations: 0 (control), 10, 32, 100, 320, and 1000mg/l

Results: All study validity criteria were met. The results are summarised in the table below.

 

Period of exposure	EL50value (with 95% confidence limits)
24 hour	561.62 mg/l (545.55-578.15 mg/l)
48 hour	354.94 mg/l (272.83-461.75 mg/l)
48 hour NOELR	100 mg/l (Steel’s Many-One Rank Test)*
48 hour 100% immobilisation	1000 mg/l

Methods used in ToxCal v5.0: Both 24-hour and 48-hour EL50’s determined by Trimmed Spearman-Karber.

*Following Shapiro-Wilk’s Test which indicated non-normal distribution. Equality of variance could not be confirmed.

All concentrations are expressed as nominal loading rates from water accommodated fractions (WAFs).