Formaldehyde, reaction products with m-phenylenediamine, sodium sulfide (Na2S) and sulfur

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:

2013-09-02 to 2013-09-05

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)

- Method:

Water Accommodated Fraction (WAF): In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items,
a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulator
y authorities in the EU and elsewhere (ECETOC 1996 and OECD 2000), is to expose the organisms to a Water Accommodated Fraction (WAF) of the
test item in cases where the test item is a complex mixture and is poorly soluble in water and in the permitted solvents. Using this approach, the
aqueous medium was prepared by mixing the test item with water for a prolonged period sufficient to ensure equilibration between the test item and
the water phase. At the completion of mixing and following a settlement period, the test item phase was separated by siphon and the test organisms
exposed to the aqueous phase, the WAF (which may contain dissolved and/or suspended and/or emulsified fractions of the test item mixture).
Exposure was expressed in terms of the original concentration of the test item in water at the start of the mixing period (loading rate) irrespective
of the actual concentration of the test item in the WAF.

Preparation of the water accommodated fraction: The water accommodated fraction (WAF) was prepared with a nominal loading of the test item of
100 mg/L as specified below. 100 mg/L of the test item were weighed out and transferred into a brown glass flask with an appropriate amount of
dilution water. This dispersion was shaken for 24 h with 20 rpm at room temperature. After a separation phase of 1 h, the aqueous phase or WAF
was removed by siphoning from the approximate centre of the glass flask to give the 100 mg/L loading rate WAF.

Limit loading rate: A WAF with a limit loading level of 100 mg/L was tested. Per definition of the WAF, all terms related to the concentration level were
given as loading level because partly dissolved compounds and mixtures cannot be related to concentrations. The WAF mentioned above is based
on the results of a non GLP preliminary range finding test conducted at the test facility under static conditions.

- Control: Dilution water without test item exposed under the same conditions as the test groups.

- Application: Per replicate, 20 g of test solution was weighed out into the test vessel. This corresponds to 20 mL per test vessel.
The daphnids were inserted with a small amount of dilution water by pipette.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna STRAUS
- Strain: Clone 5
- Source: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu)
- Breeder: DR.U.NOACK-LABORATORIEN, Kaethe-Paulus-Str. 1, 31157 Sarstedt, Germany
- Age at study initiation: 2 to 24 h old daphnids were used for the study. They were obtained by removing the parental daphnids twice within 22 h.
- Method of breeding: In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20 +/- 2°C, in an incubator, 16 h illumination,
light intensity of max. 20 µE*m-2 * s-1
- Culture medium: Elendt M4, according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L
- Culture feeding: At least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus
subspicatus, with an algae cell density of > 10^6 cells/mL
- Feeding during test: no feeding

ACCLIMATION
- Acclimation period: at least 2 h in dilution water
- Acclimation conditions (same as test or not): no
- Type and amount of food: no feeding
- Health during acclimation (any mortality observed): no abnormalities

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

Total hardness of the dilution water prior to the start of the exposure: 251 mg CaCO3/L.

Test temperature:

Temperature of the dilution water prior to the start of the exposure: 22.0°C.

pH:

pH-value of the dilution water prior to the start of the exposure: 7.98

Dissolved oxygen:

Dissolved O2-concentration of the dilution water prior to the start of the exposure: 8.51 mg/L.

Salinity:

Conductivity of the dilution water prior to the start of the exposure: 615 µS/cm.

Nominal and measured concentrations:

See "Details on test solution".

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers (4 cm ID x 7 cm H), 50 mL capacity
- Type: closed with watch glass
- Material, size, headspace, fill volume: 20 mL
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO Test water, according to OECD 202, Annex 3; pH-value: 7.8 +/- 0.2
Component Concentration [mg/L]
KCl 5.76
NaHCO3 64.8
CaCl2 * 2 H2O 294
MgSO4 * 7 H2O 123

- Water Quality Parameters at the Start of the Exposure (0 h) (measured in one additional replicate of the WAF and the control):

Nominal loading
of the water accommodated fraction (WAF) pH-value Dissolved O2-concentration
[mg/L] [mg/L]

100 7.79 8.16
Control 7.98 8.51

- Water Quality Parameters at the End of the Exposure (48 h) (measured in all replicates):

Nominal loading
of the water
accommodated fraction (WAF) pH-values Dissolved O2–concentration [mg/L]
[mg/L] Replicates Replicates
1 2 3 4 1 2 3 4

100 7.78 7.76 7.76 7.75 8.41 8.44 8.42 8.39
Control 7.60 7.69 7.71 7.78 8.45 8.34 8.46 8.57



- Culture medium different from test medium: yes
- Intervals of water quality measurement:
Dilution water: Prior to the start of the exposure (0 h), the water quality parameters (i.e. pH value, dissolved oxygen concentration, temperature,
conductivity and total hardness) of the dilution water were measured.
Test media: At the start of the exposure (0 h), the water quality parameters of the fresh media (i.e. pH value, dissolved oxygen concentration)
were measured in one additional replicate of the WAF and the control. At the end of the exposure (48 h), the water parameters of the old media
were measured in all replicates of the WAF and the control.
Temperature: The incubator temperature was recorded throughout the period of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16/8 light/dark cycle
- Light intensity: Diffuse light, light intensity of max. 20 µE*m^-2 * s^-1
- Temperature: 18 - 22°C, constant within +/- 1°C

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Immobilisation was determined in all groups after 24 and 48 h. An animal was considered to be immobile, if it was not able to swim in the water
phase within 15 seconds after gentle agitation of the test vessel.

TEST CONCENTRATIONS
- Range finding study: A preliminary test was carried out with two water accommodated fractions of the test item C.I. LEUCO SULPHUR BROWN 37
(CAS: 70892-34-1) "Press-cake" with nominal loadings of nominal 10.0 and 100 mg/L under static conditions over a period of 48 h. No effects were
observed on Daphnia magna after an exposure period of 48 h. The results are presented in the table below.

Immobilisation Rates in the non GLP Preliminary Test (n = 20, divided into 2 replicates with 10 daphnids each):

Nominal loading Immobilisation [%]
of the water accommodated fraction (WAF) 24 h 48 h
[mg/L] Replicates Replicates
1 2 MV 1 2 MV
100 10 10 1 0 0 0 0
10.0 0 0 0 0 0 0
Control 0 0 0 0 0 0

MV = mean value

Reference substance (positive control):

yes

Duration:

48 h

Dose descriptor:

other: EL10, EL50, EL100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: A WAF with a limit loading level of 100 mg/L was tested. Per definition of the WAF, all terms related to the concentration level were given as loading level because partly dissolved compounds and mixtures cannot be related to concentrations.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: see EL10, EL50, EL100

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: see EL10, EL50, EL100

Basis for effect:

mobility

Details on results:

- The percentage immobility, determined at the water accommodated fraction with a nominal loading of 100 mg/L of the test item
C.I. LEUCO SULPHUR BROWN 37 (CAS: 70892-34-1) "Press-cake" and the control after 24 and 48 h under static conditions, is presented in the table
below. No effects were observed at the WAF and the control group.

Immobilisation Rates of the Definitive Test after 24 and 48 hours of Exposure (n = 20, divided into 4 replicates with 5 daphnids each):

Nominal loading IMMOBILISATION [%]
of the water
accommodated fraction (WAF) 24 h 48 h
[mg/L] Replicates Replicates

1. 2. 3. 4. MV 1. 2. 3. 4. MV
100 0 0 0 0 0 0 0 0 0 0
Control 0 0 0 0 0 0 0 0 0 0

EL10 / 50 / 100 > 100 mg/L > 100 mg/L
NOEL 100 mg/L 100 mg/L
LOEL > 100 mg/L > 100 mg/L


The WAF with a nominal loading of 100 mg/L of the test item was slightly brown and clear at the start of the exposure. The test item precipitated
partly after 24 h.

Results with reference substance (positive control):

- Results with reference substance valid? yes

The percentage immobility for the reference item was determined after 24 h. The EC50-value with 95 % confidence interval (CI) was calculated by sigmoidal dose-response regression. The EC50-value for the most recent of the monthly performed reference tests was: EC50: 1.69 mg/L (CI 1.65 - 1.72 mg/L). The EC50-value of the reference item potassium dichromate after 24 h is within the prescribed concentration range of 0.6 - 2.4 mg/L of quality criteria according to AQS P 9/2 (02/2000) for daphnids clone 5 cultured in Elendt M4 medium. The EC50-value of the reference item is also within the recommended range of 0.6 - 2.1 mg/L according to OECD-Guideline 202.

Reported statistics and error estimates:

- The effects of the WAF were deduced directly from the observed immobilisation rates.

- An EC50-value was calculated only for the reference item by sigmoidal dose-response regression. The confidence interval for EC50 was calculated from the best-fit values, the standard error and the t-distribution with the software GraphPad prism5.

Validity criteria fulfilled:

yes

Conclusions:

At the water accommodated fraction (WAF) with a nominal loading of 100 mg/L of the test item C.I. LEUCO SULPHUR BROWN 37 (CAS: 70892-34-1), no effects were observed on Daphnia magna.

Executive summary:

 

In the acute immobilisation test with Daphnia magna (STRAUS), the effects of the Water Accommodated Fraction (WAF)* with the nominal loading of 100 mg/L of the test item C.I. LEUCO SULPHURBROWN 37 (CAS: 70892-34-1) "Press-cake"(batch number: ESA0010188) were determined according to OECD 202 (2004).The limit test was conducted under static conditions over a period of 48 hours from 2013-09-02 to 2013-09-05, with the definitive exposure phase from 2013-09-03 to 2013-09-05, at Dr. U. Noack-Laboratorien, Käthe-Paulus-Str. 1,31157, Germany.

The test item is a black, wet solid and insoluble in water. In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996 and OECD 2000), is to expose the organisms to a Water Accommodated Fraction (WAF) of the test item in cases where the test item is a complex mixture and is poorly soluble in water and in the permitted solvents.

Using this approach, the aqueous medium was prepared by shaking the test item with dilution water at 20 rpm for 24 hours at room temperature to ensure equilibration between the test item and the water phase.100 mg/L of the test item were weighed out and transferred into a brown glass flask with an appropriate amount of dilution water. After the completion of shaking and following a 1-h settlement period, the test item phase was separated by siphonand the test organisms exposed to the clear aqueous phase, the WAF. Twenty daphnids were exposed to the WAF and the control.

No analytical evaluation of the WAF and the control was carried out, because the test item is insoluble in water and other solvents, which are compatible with analytical systems. Exposures are expressed in terms of the original concentrations of the test item C.I. LEUCO SULPHURBROWN 37 (CAS: 70892-34-1) "Press-cake" in water at the start of the mixing period (loading rate), irrespective of the actual concentrations of the test item in the WAFs.

Water quality parameters (i.e. pH-value and dissolved oxygen concentration), measured at the start (0 h) and at the end of the exposure (48 h), were within the acceptable limits. The validity criteria of the test guidelines were fulfilled.

 

At the water accommodated fraction (WAF)* with a nominal loading of 100 mg/L of the test item C.I. LEUCO SULPHURBROWN 37 (CAS: 70892-34-1), no effects were observed on Daphnia magna.

Description of key information

At the water accommodated fraction (WAF) with a nominal loading of 100 mg/L of the test item  no effects were observed on Daphnia magna.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

100 mg/L

Additional information

Two acute immobilisation tests with daphnia magna (STRAUS) according OECD Guideline for Testing of Chemicals, No 202 (2004) are available. Due to the low water solubility of the test item, a dispersion of the test item with the loading rate of 100 mg/L was ultrasonic treated for 15 minutes in the first test and continuously stirred at room temperature in the dark over 96 hours. Then, the dispersion was filtered. The undiluted filtrate with the loading rate of 100 mg/L and dilutions 1:2, 1:4, 1:8 and 1:16 of the undiluted filtrate with the loading rate of 100 mg/L were used as test media. This corresponds to theoretical concentrations of 100, 50, 25, 12.5 and 6.3 mg/L. Additionally, a control was tested in parallel.

 

In the second test the preparation of aqueous media was different. In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996 and OECD 2000), is to expose the organisms to a Water Accommodated Fraction (WAF) of the test item in cases where the test item is a complex mixture and is poorly soluble in water and in the permitted solvents.

Using this approach, the aqueous medium was prepared by shaking the test item with dilution water at 20 rpm for 24 hours at room temperature to ensure equilibration between the test item and the water phase.100 mg/L of the test item were weighed out and transferred into a brown glass flask with an appropriate amount of dilution water. After the completion of shaking and following a 1-h settlement period, the test item phase was separated by siphon and the test organisms exposed to the clear aqueous phase, the WAF. Twenty daphnids were exposed to the WAF and the control.

 

In the first test an EC50 of 48 mg/L is reported, in the second test no effects were observed on Daphnia magna at the WAF with nominal loading of 100 mg/L of the test item. The effect shown in the first report probably based on the different method of preparing the Water Accommodated Fraction (WAF).