2,4,6-triisopropyl-m-phenylene diisocyanate

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-06-26 - 2020-08-12 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.20 (Daphnia magna Reproduction Test)

Version / remarks:

Council Regulation (EC) No 440/2008, Method C.20 'Daphnia Magna, Reproduction Test' (2008)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

OECD Guideline No. 211 "Daphnia Magna Reproduction Test" (adopted: 2nd October 2012)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Guidance Document on aqueous-phase Aquatic Toxicity testing of Difficult Test Chemicals No. 23 (2019)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: nominal 105, 34, 11, 3.4, 1.1 and 0.34 µg/L and control
- Sampling method: 0h samples were taken from the freshley prepared media and 48h samples were taken from the test vessels at test medium renewal.
Control: on day 4, 12 and 18 in the freshly prepared media (0h); on day 6, 14 and 20 in the aged media (48h)
Test concentrations: all test concentrations on day 4, 12 and 18 in the freshly prepared media (0h); on day 6, 14 and 20 in the aged media (48h)
- Sample storage conditions before analysis: The samples were analysed immediately after sampling.

Vehicle:

yes

Remarks:

dimethylformamid (DMF) at a final test medium concentration of 100 µL/L

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: To bring the test item into solution and to enable the preparation of reproducible test concentrations throughout the test, dimethylformamide (DMF) was used as solvent carrier. 5.25 mg of the test item was weighed and was dissolved in 5 mL DMF. 0.5 mL of this solution were taken and were filled with dilution water to a volume of 5000 mL. This solution was treated in an ultrasonic bath for 0,5 hours and was then gently stirred for 20 hours on a magnetic stirrer. The pH was measured and ranged between 7.9 and 8.0 and was not further adjusted. To produce the different test item concentrations appropriate amounts of the stock solution were diluted with dilution water which contains DMF in an amount such that the final concentration is 0.1 mL solvent per liter, to a volume of 2000 mL. The test media were prepared just before introduction of the daphnids (= start of the test) and each test medium renewal.
- Controls: Control (dilution water only), solvent control (dilution water and vehicle)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): dimethylformamide (DMF)
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 100 µL/L

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: clone of the Federal Health Office (BGA) in Berlin
- Age at study initiation (mean and range, SD): less than 24 hours
- Stage and instar at study initiation: neonates
- Method of breeding: A population of parthenogenetic females of synchronized age structure has been maintained for several years in the test facility under constant temperature conditions (20 +/- 2 °C) at a 16 : 8 hour light-dark photoperiod (light intensity:
< 15-20 µE x m-2 x s-1). The culture water (so-called 'M4 medium') was partly renewed once a week. The Daphnia were exclusively fed unicellular green algae (Desmodesmus subspicatus) 'ad libitum'. Mortalities of parent Daphnia during the culture period were recorded daily in a semi-quantitative way.
- Feeding during test
- Food type: Living Desmodesmus subspicatus from batch culture
- Amount: day 0 - 7: 0.1 mg C / Daphnia / day and day 8 – 21: 0.2 mg C / Daphnia / day
- Frequency: The test Daphnia were fed daily.

ACCLIMATION
- Acclimation period: Was not necessary, since the test was performed in the same medium as the culturing.

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES, INCLUDING CULTURING CONDITIONS:
The neonates were separated from their parent Daphnia by filtration prior to the test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

21 d

Hardness:

Water hardness was above the recommended value of 140 mg/L (as CaCO3).

Test temperature:

19.6 - 20.7 °C in the freshly prepared media
20.2 - 20.8 in the aged media

pH:

7.8 - 8.2 in the freshly prepared media
7.7 - 8.0 in the aged media

Dissolved oxygen:

8.2 - 10.0 mg/L in the freshly prepared media
8.4 - 9.3 mg/L in the aged media

Nominal and measured concentrations:

nominal: 105, 34, 11, 3.4, 1.1 and 0.34 µg/L, control and solvent control
geometric mean measured: 74.2, 14.9, 8.53 and 0.704 µg/L, control and solvent control; no time weighted mean measured concentration was determined for the lowest test concentrations of 0.34 and 0.0011 µg/L, as measured concentrations were below the limit of quantification of the HPLC-MS method accompanying the test. Furthermore the concentrations were below the determined 21d NOEC and do not have an influence on the outcome of the study.

Details on test conditions:

TEST SYSTEM
- Test vessel: 150 mL glass beakers
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 100 mL of test medium
- Aeration: none
- Renewal rate of test solution (frequency/flow rate): The test media of all test concentrations, the solvent control and the control were renewed every 48 hours.
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- No. of vessels per vehicle control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4 medium according to OECD 211
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light / 8 hours dark
- Light intensity: not exceeding 15-20 uE/m2-s which corresponds to 1000- 1300 Iux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Observations on mortality of adults and offspring occurrence were made daily. The number of offspring was counted at each water renewal and determined for each day. Dead offspring was counted if possible at each water renewal. Dead adults and offspring were removed at each water renewal day. The date of first eggs, of first brood and the number of offspring per adult in each treatment replicate was documented. Additionally, any signs of intoxication (e.g. pale adults, aborted eggs) were recorded.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.1
- Range finding study
- Results used to determine the conditions for the definitive study: Several pre-experiments were performed to determine the solubility and the reproducibility of test medium concentrations. Further pre-experiments were performed to determine a suitable concentration range and to establish suitable analytical methods to prepare the test solutions.The pre-experiments were not performed in compliance with the GLP-Regulations.

Reference substance (positive control):

not required

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 74.2 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

> 74.2 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 74.2 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 74.2 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

> 74.2 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 74.2 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 74.2 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality: Number of living offspring produced per surviving parental animal {for Daphnia magna, TG 211}

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

> 74.2 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality: Number of living offspring produced per surviving parental animal {for Daphnia magna, TG 211}

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 74.2 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality: Number of living offspring produced per surviving parental animal {for Daphnia magna, TG 211}

Details on results:

- Behavioural abnormalities: No sublethal effects or behavioural abnormalities were recorded for the tretament groups and the control groups.
- Mortality of control: no
- Other adverse effects control: no
- Immobilisation of control: no
- Abnormal responses: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no

Reported statistics and error estimates:

All values were calculated using the statistic programme ToxRatPro Version 2.10 (released 2010-09-10). As there is no statistically significant difference between Control and DMF Control, the control was excluded and the DMF control was used for statistical calculations.

Validity criteria fulfilled:

yes

Conclusions:

The study was conducted under GLP according to EU Method C.20 (2018), which is in the relevant parts equivalent to OECD Guideline No. 211 (adopted: 2nd October 2012) on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or deviations from the guidelines, the validity criteria were met. Hence, the results can be considered as reliable to assess the long-term toxicity of the test substance towards daphnia.
In a semi-static test neonates (< 24 h) of the freshwater invertebrate Daphnia magna were exposed to the test substance at the concentrations of nominal 105, 34, 11, 3.4, 1.1 and 0.34 µg/L (each in 100 µL DMF/L), a solvent control (100 µL DMF/L) and a control for a period of 21 days under defined conditions. Using HPLC MS/MS detection time weighted average concentrations of 74.2, 15.0, 8.53 and 0.704 µg test item/L were determined for the highest four test concentrations. Based on the obtained results no significant effects against daphnia were observed up to the highest concentration tested. Thus, the 21 d NOEC was determined to be nominal 105 µg/L, corresponding to 74.2 µg test item/L based on geometric mean measured concentrations, for the observed effect parameters mortality of adults and number of offspring per introduced adult and per survived adult. The test concentration of nominal 105 µg/L represents the highest achievable and reproducible concentration of the registered test substance for the performance of an aquatic long-term study.

Executive summary:

The purpose of this study was to evaluate the influence of the test material on mortality and reproduction of Daphnia magna during an exposure period of 21 days. Therefore, female Daphnia (< 24 hours old) taken from an in-house laboratory culture of the test facility, were exposed in a semi-static test to aqueous test media containing the test item at concentrations of nominal 105, 34, 11, 3.4, 1.1 and 0.34 µg test item/L (each in 100 µL DMF/L) ), a solvent control (100 µL DMF/L) and a control. Using HPLC MS/MS detection time weighted average concentrations of 74.2, 15.0, 8.53 and 0.704 µg test item/L were determined for the highest four test concentrations. No time weighted mean measured concentration was determined for the lowest test concentrations of 0.34 and 1.1 µg/L, as measured concentrations were below the limit of quantification of the HPLC-MS method accompanying the test. Furthermore the concentrations were below the determined 21d NOEC and do not have an influence on the outcome of the study.

The test animals were exposed to the test media for a period of 21 days under defined conditions. Following several independent pre-experiments to determine the solubility and stability of the test material in the test medium and the need to develop a reproducible analytical method, dimethylformamide (DMF) was used as solvent additive to achieve stable and reproducible concentrations in the test medium. The mortality of adults, the number of offspring per introduced adult and the number of offspring per surviving adult were compared with corresponding parameters in the controls.

Based on the obtained results no toxic effects against daphnia were observed up to the highest concentration of nominal 105 µg/L, corresponding to the time weighted average concentration of 74.2 µg/L, under exposure conditions. As analytical recoveries in the test media samples were not within 80 -120 % of nominal, the following endpoints were determined referring to the time weighted average concentrations of the test material:

EC₅₀ (21d) mortality > 74.2 µg/L

EC₁₀ (21d) mortality > 74.2 µg/L

NOEC (21d) mortality ≥ 74.2 µg/L

 

EC₅₀ (21d) reproduction per introduced adult > 74.2 µg/L

EC₁₀ (21d) reproduction per introduced adult > 74.2 µg/L

NOEC (21d) reproduction per introduced adult ≥ 74.2 µg/L

 

EC₅₀ (21d) reproduction per surviving adult > 74.2 µg/L

EC₁₀ (21d) reproduction per surviving adult > 74.2 µg/L

NOEC (21d) reproduction per surviving adult ≥ 74.2 µg/L

Reference 2

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

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

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study with GLP compliance.

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

GLP compliance:

yes

Remarks:

No further details available.

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
- Molecular formula: C9H6N2O2
- Molecular weight: 174.15 g/mol
- Smiles notation: CC(C)c1c(N=C(=O))c(C(C)C)c(N=C(=O))c(C(C)C)c1
- Substance type: aromatic diisocyanate

Analytical monitoring:

yes

Details on sampling:

No details available.

Vehicle:

no

Details on test solutions:

No details available.

Test organisms (species):

Daphnia magna

Details on test organisms:

- Female Daphnia
- Age: less than 24 hours.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

No post exposure observation period reported.

Hardness:

206 - 281 mg/L as CaCO3

Test temperature:

20.6 - 21.0 °C

pH:

No details available.

Dissolved oxygen:

No details available.

Salinity:

Not applicable.

Nominal and measured concentrations:

0.28, 0.55, 1.10, 2.20 and 4.40 mg/L

Details on test conditions:

At the end of the test, the total number of living offspring produced per parent animal alive at the end of the test is assessed and compared to controls.
Since the test substance readily hydrolyzes to give polyureas, oligoureas, 2,4-TDA, and 2,6-TDA, the TDA concentration was analyzed once per week for each test solution, on the fresh test solutions, and on the same test solutions after Daphnia exposure at renewal.

Reference substance (positive control):

yes

Remarks:

No details available.

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

2 mg/L

Nominal / measured:

nominal

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1.1 mg/L

Nominal / measured:

nominal

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

2.2 mg/L

Nominal / measured:

nominal

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

other: NOEC (TDA)

Effect conc.:

0.5 mg/L

Nominal / measured:

meas. (not specified)

Basis for effect:

reproduction

Details on results:

Conversion of TDI to TDA was incomplete in all the experiments, with yields shown to be between 57 and 69% in the initial test solutions at the beginning of the tests. The TDA analysis figures indicate a derived NOEC for TDA of 0.5 mg/L.

Results with reference substance (positive control):

No details available.

Reported statistics and error estimates:

No details available.

Validity criteria fulfilled:

yes

Remarks:

Basic criteria were met.

Conclusions:

The study is regarded as a valid guideline study with GLP compliance. According to the reported NOEC (1.1 mg/L) the test substance is not classified as chronic toxic to aquatic invertebrates.

Executive summary:

The chronic toxicity of m-tolylidene diisocyanate to Daphnia magna was investigated according to OECD Guideline 211 for a test duration of 21 days (Cerbelaud et.al., 1997). Young female daphnids (less than 24 hours old) were exposed to nominal test concentrations of 0.28, 0.55, 1.10, 2.20 and 4.40 mg/L. Analysis of 2,4- and 2,6-TDA was carried out using High Performance Liquid Chromatography (HPLC). The NOEC for the test substance was defined as 1.1 mg/L. Conversion of TDI to TDA was incomplete in all the experiments, with yields shown to be between 57 and 69% in the initial test solutions at the beginning of the tests. The TDA analysis indicates a derived NOEC for TDA of 0.5 mg/L.

Reference 3

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

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

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Non-GLP guideline study with acceptable restrictions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

Cited as OECD Guide-line 202, part 2 (Daphnia sp., Reproduction Test)

GLP compliance:

no

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
- Molecular formula: C9H6N2O2
- Molecular weight: 174.15 g/mol
- Smiles notation: CC(C)c1c(N=C(=O))c(C(C)C)c(N=C(=O))c(C(C)C)c1
- Substance type: aromatic diisocyanate

Analytical monitoring:

no

Details on sampling:

Not applicable.

Vehicle:

no

Details on test solutions:

No details available.

Test organisms (species):

Daphnia magna

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

21 d

Hardness:

about 250 mg CaCO3/L

Test temperature:

20 °C

pH:

No details available.

Dissolved oxygen:

No details available.

Salinity:

Not applicable.

Nominal and measured concentrations:

0.05, 0.16 and 0.50 mg/L

Details on test conditions:

- Test material preparation: magnetically stirring into the medium for 1 h prior to sampling.
- Photoperiod: 12 h.
- Test medium renewal: thrice weekly.
At each renewal of the test medium, activated sludge extract from a domestic sewage treatment plant (final COD concentration in the test water: 5 mg/L) was added to assure maximum reproduction rates under control conditions.

Reference substance (positive control):

not specified

Remarks:

Not stringent required under OECD 211.

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

>= 0.5 mg/L

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.5 mg/L

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

>= 0.5 mg/L

Basis for effect:

reproduction

Details on results:

No effect observed at highest concentration tested. Due to the rapid hydrolysis of TDI this test essentially measured the toxicity of hydrolysis products. Preparations were found to be heterogeneous with much insoluble product.

Results with reference substance (positive control):

Not applicable.

Reported statistics and error estimates:

No details available.

Validity criteria fulfilled:

yes

Remarks:

Basic criteria were met.

Conclusions:

The study is regarded as a valid guideline study without GLP compliance. According to the reported NOEC (> 0.50 mg/L) the test substance is probably not classified as chronic toxic to aquatic invertebrates.

Executive summary:

The chronic toxicity of m-tolylidene diisocyanate to Daphnia magna was investigated according to OECD Guideline 211 for a test duration of 21 days (Caspers et.al., 1986). The test material was stirred into the medium for 1 h prior to sampling and the medium was renewed thrice weekly. At each renewal of the test medium, activated sludge extract from a domestic sewage treatment plant (final COD concentration in the test water: 5 mg/L) was added to assure maximum reproduction rates under control conditions. This limit test was conducted with test concentrations of 0.05, 0.16 and 0.50 mg/L. No effect observed at highest concentration tested, therefore the EC50, LOEC and NOEC values were determined as greater or equal to 0.50 mg/L. Due to the rapid hydrolysis of TDI this test essentially measured the toxicity of hydrolysis products. Preparations were found to be heterogeneous with much insoluble product.

Description of key information

1) Key_Long-term toxicity to aquatic invertebrates: NOEC (21d) ≥ 74.2 µg/L (time weighted average); EC₁₀ (21d) > 74.2 µg/L (time weighted average) for Daphnia magna based on mortality, reproduction per introduced adult and reproduction per surviving adult (semi-static, freshwater, OECD 211, GLP)

2) Supporting_Long-term toxicity to aquatic invertebrates_RA TDI: NOEC (21d) = 1.1 mg/L (nominal) for Daphnia magna (static, freshwater, OECD 211, GLP)

3) Supporting_Long-term toxicity to aquatic invertebrates_RA TDI: NOEC (21d) = 0.5 mg/L (nominal) for Daphnia magna (semi-static, freshwater, OECD 211, non-GLP)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Effect concentration:

>= 74.2 µg/L

Additional information

The purpose of this study was to evaluate the influence of the test material on mortality and reproduction of Daphnia magna during an exposure period of 21 days. Therefore, female Daphnia (< 24 hours old) taken from an in-house laboratory culture of the test facility, were exposed in a semi-static test to aqueous test media containing the test item at concentrations of nominal 105, 34, 11, 3.4, 1.1 and 0.34 µg test item/L (each in 100 µL DMF/L) ), a solvent control (100 µL DMF/L) and a control. Using HPLC MS/MS detection time weighted average concentrations of 74.2, 15.0, 8.53 and 0.704 µg test item/L were determined for the highest four test concentrations. No time weighted mean measured concentration was determined for the lowest test concentrations of 0.34 and 1.1 µg/L, as measured concentrations were below the limit of quantification of the HPLC-MS method accompanying the test. Furthermore the concentrations were below the determined 21d NOEC and do not have an influence on the outcome of the study.

The test animals were exposed to the test media for a period of 21 days under defined conditions. Following several independent pre-experiments to determine the solubility and stability of the test material in the test medium and the need to develop a reproducible analytical method, dimethylformamide (DMF) was used as solvent additive to achieve stable and reproducible concentrations in the test medium. The mortality of adults, the number of offspring per introduced adult and the number of offspring per surviving adult were compared with corresponding parameters in the controls.

Based on the obtained results no toxic effects against daphnia were observed up to the highest concentration of nominal 105 µg/L, corresponding to the time weighted average concentration of 74.2 µg/L, under exposure conditions. As analytical recoveries in the test media samples were not within 80 -120 % of nominal, the following endpoints were determined referring to the time weighted average concentrations of the test material:

EC₅₀(21d) mortality > 74.2 µg/L

EC₁₀(21d) mortality > 74.2 µg/L

NOEC (21d) mortality≥74.2 µg/L

 

EC₅₀(21d) reproduction per introduced adult > 74.2 µg/L

EC₁₀(21d) reproduction per introduced adult > 74.2 µg/L

NOEC (21d) reproduction per introduced adult≥74.2 µg/L

 

EC₅₀(21d) reproduction per surviving adult > 74.2 µg/L

EC₁₀(21d) reproduction per surviving adult > 74.2 µg/L

NOEC (21d) reproduction per surviving adult≥74.2 µg/L

These results are supported by two experiments on the chronic toxicity of the read-across substance m-tolylidene diisocyanate (TDI) to aquatic invertebrates conducted with Daphnia magna according to OECD Guideline 211.

In the supporting study performed by Cerbelaud et.al. (1997), young female daphnids (less than 24 hours old) were exposed to nominal test concentrations of 0.28, 0.55, 1.10, 2.20 and 4.40 mg/L for a test duration of 21 days. Analysis of 2,4- and 2,6-TDA was carried out using High Performance Liquid Chromatography (HPLC). The NOEC for the test substance was defined as 1.1 mg/L. Conversion of TDI to TDA was incomplete in all the experiments, with yields shown to be between 57 and 69% in the initial test solutions at the beginning of the tests. The TDA analysis indicates a derived NOEC for TDA of 0.5 mg/L.

In the second supporting study, the test material was stirred into the medium for 1 h prior to sampling and the medium was renewed twice weekly (Caspers et al., 1986). At each renewal of the test medium, activated sludge extract from a domestic sewage treatment plant (final COD concentration in the test water: 5 mg/L) was added to assure maximum reproduction rates under control conditions. This test was conducted with test concentrations of nominal 0.05, 0.16 and 0.50 mg/L. No effect observed at highest concentration tested, therefore the EC₅₀, LOEC and NOEC values were determined as greater or equal to nominal 0.50 mg/L. Due to the rapid hydrolysis of TDI this test essentially measured the toxicity of hydrolysis products. Preparations were found to be heterogeneous with much insoluble product.