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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 Aug 2010 - 05 Apr 2011
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)
Remarks:
Swiss Federal Office of Public Health, Consumer protection directorate, Notification authority for chemicals, Bern, Switzerland
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 62.5, 125, 250, 500 and 1000 mg/L
- Sampling method: Aliquots of the freshly prepared test solutions of the control and all treatment levels were sampled at hour 0. The aged test solutions were sampled at hour 48. Three quality control (QC) samples fortified with AEPD and a blank control were prepared at each sampling interval at nominal concentrations approximating the test concentrations and remained with the exposure solution sample throughout the analytical process. Analysis of the QC samples was used to judge the precision and quality control maintained during the analytical process.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: For both definitive tests a primary stock solution with a concentration of 1000 mg test item/L was prepared prior to test initiation by dissolving 2.0018 g of the test item with 2000 mL dilution water. Different stock concentrations and volumes of dilution water (Elendt M4 medium) were then used to achieve desired nominal test concentrations.
- Eluate: no
- Controls: negative control, containing only dilution water
- Evidence of undissolved material: All resulting test solutions were shaken by hand for 30 seconds and observed to be clear and colorless, with no visible undissolved test item.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Source: originally obtained from Springborn Smithers Laboratories Inc. (new Smithers Viscient), Wareham, USA
- Age at study initiation: < 24 hours
- Feeding during test: none

ACCLIMATION
- Acclimation period: Prior to testing, the daphnids were held in glass beakers under a photoperiod of 16 hours light and 8 hours darkness with a 30 minute transition period.
- Acclimation conditions (same as test or not): same as test
- Type and amount of food: 2.0 mL of a solution containing approximately 4 x 107 cells/mL of the unicellular green alga, Ankistrodesmus falcatus (ANK) and approximately four drops of a combination of yeast, cereal leaves and flaked fish food (YCT)
- Feeding frequency: daily
- Health during acclimation (any mortality observed): not reported
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
Definitive Test 1 (no pH adjustment performed): Total hardness of dilution water as CaCO3: 140 mg/L
Definitive Test 2 (pH adjustment performed): Total hardness of dilution water as CaCO3: 156 mg/L
Test temperature:
Definitive Test 1 (no pH adjustment performed): 19 to 20 °C
Definitive Test 2 (pH adjustment performed): 20 to 21 ºC
pH:
Definitive Test 1 (no pH adjustment performed): 7.13 to 10.26
Definitive Test 2 (pH adjustment performed): 7.77 to 8.96
Dissolved oxygen:
Definitive Test 1 (no pH adjustment performed): 7.30 to 8.48 mg/L
Definitive Test 2 (pH adjustment performed): 7.81 to 9.44 mg/L
Nominal and measured concentrations:
nominal: 0, 62.5, 125, 250, 500 and 1000 mg/L
measured: 0, 53.0, 108, 209, 444 and 931 mg/L (Definitive Test 1, no pH adjustment performed)
measured: 0, 53.8, 111, 205, 409 and 859 mg/L (Definitive Test 2, pH adjustment performed)
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type: closed (loosely covered with a glass plate)
- Material, size, headspace, fill volume: 250 mL glass beakers, each containing 200 mL of test solution
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Alkalinity: 26 mg/L as CaCO3 (Definitive Test 1, no pH adjustment performed); 36 mg/L as CaCO3 (Definitive Test 2, pH adjustment performed)
- Conductivity: 550 μS/cm (Definitive Test 1, no pH adjustment performed); 360 μS/cm (Definitive Test 2, pH adjustment performed)
- Intervals of water quality measurement: pH, dissolved oxygen concentration at 0, 24 and 48 hours; temperature continuously in adjacent vessel

OTHER TEST CONDITIONS
- Adjustment of pH: yes, in Definitive Test 2
- Photoperiod: 16-hour light, 8-hour dark
- Light intensity: 228 to 342 (Definitive Test 1), 240 to 304 Lux (Definitive Test 2), using fluorescent bulbs

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

TEST CONCENTRATIONS
- Spacing factor for test concentrations: x2
- Range finding study
- Test concentrations: control, 0.10, 1.0, 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: Following 48 hours of exposure, 100% immobilization was observed in the 1000 mg/L treatment level. No immobilization was observed among daphnids exposed to the control and 0.10, 1.0, 10 and 100 mg/L treatment levels.
Based on these results definitive test concentrations of 6.25, 125, 250, 500 and 1000 mg/L and a control were selected for the definitive exposure.
Reference substance (positive control):
no
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
558 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CL: 413 - 667; Definitive Test 1 (no pH adjustment performed)
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
374 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CL: 344 - 414; Definitive Test 1 (no pH adjustment performed)
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
209 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: Definitive Test 1 (no pH adjustment performed)
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 859 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: Definitive Test 2 (pH adjustment performed)
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
668 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CL: 645 - 679; Definitive Test 2 (pH adjustment performed)
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
205 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: Definitive Test 2 (pH adjustment performed)
Details on results:
- Behavioural abnormalities:
Definitive Test 1 (No pH Adjustment Performed): After 24 hours of exposure, immobilization of 0, 0, 0, 0, 45 and 100% were observed in the dilution water control and the 53.0, 108, 209, 444 and 931 mg test item/L treatment levels, respectively. Following 48 hours of exposure, immobilization of 5, 0, 0, 5, 85 and 100% were observed in the dilution water control and the 53.0, 108, 209, 444 and 931 mg test item/L treatment levels, respectively. One daphnid was observed to carry adhered particles in the 444 mg test item/L treatment level at hour 48.
Definitive Test 2 (pH Adjustment Performed): After 24 hours of exposure, immobilization of 0, 0, 0, 0, 5 and 35% were observed in the dilution water control and the 53.8, 111, 205, 409 and 859 mg test item/L treatment levels, respectively. Following 48 hours of exposure, immobilization of 0, 0, 0, 0, 5 and 100% were observed in the dilution water control and the 53.8, 111, 205, 409 and 859 mg test item/L treatment levels, respectively. After 24 hours 1 lethargic daphnid was observed at a treatment level of 53.8 mg test item/L. Since no effect was observed at the 111 and 205 mg test item/L, the lethargic daphnid was not considered an effect of the test item. At 409 and 859 mg test item/L, 3 and 10 lethargic daphnids were observed after 24 hours, respectively. One lethargic daphnid at the 859 mg test item/L test concentration showed in addition erratic behaviour. Five lethargic, 1 erratic and 1 floater were observed in the 409 mg test item/L treatment level at hour 48.

Based on the behaviour of the test item in daphnid medium and the impact on the pH of the solution (increase) a second exposure was performed using pH adjustments (pH 8.0 ± 0.1) of the exposure solutions. The results of the second exposure should be considered more robust than those of the first exposure. The exposure conditions provided during the second test are more conclusive to daphnid behaviour and it is clear that the pH shift impacted the observed endpoints in the first exposure.

Table 1: Definitive Test 1 (No pH Adjustment Performed): Corresponding immobilization and observations made during the 48-hour toxicity test exposing daphnids (Daphnia magna) to AEPD:

Geometric Mean Measured Concentration (mg test item/L)

Cumulative percent of immobilized Daphnia (a)

24-Hour

48-Hour

A

B

C

D

Mean

A

B

C

D

Mean

Control

0

0

0

0

0

0

0

0

20

5

 

(0)

(0)

(0)

(0)

 

(0)

(0)

(0)

(1)

 

53.0

0

0

0

0

0

0

0

0

0

0

 

(0)

(0)

(0)

(0)

 

(0)

(0)

(0)

(0)

 

108

0

0

0

0

0

0

0

0

0

0

 

(0)

(0)

(0)

(0)

 

(0)

(0)

(0)

(0)

 

209

0

0

0

0

0

0

20

0

0

5

 

(0)

(0)

(0)

(0)

 

(0)

(1)

(0)

(0)

 

444

40

80

40

20

45

100

100

80

60

85

 

(2)

(4)

(2)

(1)

 

(5)

(5)

(4)

(3)(b)

 

931

100

100

100

100

100

100

100

100

100

100

 

(5)

(5)

(5)

(5)

 

(5)

(5)

(5)

(5)

 

(a) The actual number of immobilized daphnids is presented in parentheses.

(b) Sublethal effect (1 daphnid carrying adhered particles) was observed.

Table 2: Definitive Test 2 (pH Adjustment Performed): Corresponding immobilization and observations made during the 48-hour toxicity test exposing daphnids (Daphnia magna) to AEPD.

Geometric Mean Measured Concentration (mg test item/L)

Cumulative percent of immobilized Daphnia (a)

24-Hour

48-Hour

A

B

C

D

Mean

A

B

C

D

Mean

Control

0

0

0

0

0

0

0

0

0

0

 

(0)

(0)

(0)

(0)

 

(0)

(0)

(0)

(0)

 

53.0

0

0

0

0

0

0

0

0

0

0

 

(0)

(0)

(0)

(0)

 

(0)

(0)

(0)

(0)

 

111

0

0

0

0

0

0

0

0

0

0

 

(0)

(0)

(0)

(0)

 

(0)

(0)

(0)

(0)

 

205

0

0

0

0

0

0

0

0

0

0

 

(0)

(0)

(0)

(0)

 

(0)

(0)

(0)

(0)

 

409

20

0

0

0

5

0

0

0

0

0

 

(1)

(0)(b)

(0)(b)

(0)(b)

 

(0)(b)

(0)

(0)(b,c)

(0)(b,d)

 

859

60

20

20

40

35

0

0

0

0

0

 

(3)(b)

(1)(b,c)

(1)(b)

(2)(b)

 

(0)

(0)

(0)

(0)

 

(a) The actual number of immobilized daphnids is presented in parentheses.

(b) Sublethal effect (lethargic daphnid(s)) was observed.

(c) Sublethal effect (erratic daphnid(s)) was observed.

(d) Sublethal effect (floater) was observed.

Description of key information

EC50(48h) = 668 mg/L for Daphnia magna (OECD 202)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
668 mg/L

Additional information

There is one short-term aquatic invertebrate study available for 2-amino-2-ethyl-1,3-propanediol (AEDP), which was used as key study for the assessment. The study was conducted according the OECD guideline 202 and GLP (Hoffmann, 2011). The test organism Daphnia magna was exposed to AEPD in a static system for 48 hours, at the nominal test concentrations 0, 62.5, 125, 250, 500 and 1000 mg/L. The test was performed with and without pH adjustment, leading to EC50 values of 668 mg/L and 374 mg/L, respectively. Based on this data, pH had an effect on the immobilisation of daphnids, and the value obtained with pH adjustment should be used for the assessment of toxicity.