Registration Dossier

Administrative data

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-12-14 to 2009-12-18
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Phosphorpentoxid
- Molecular formula: P4O10
- Molecular weight: 283.9 g/mol
- Physical state: Solid, white powder
- Analytical purity: 99.7 % (m/m)
- Purity test date: 2009-09-23
- Lot/batch No.: DEH2 018965
- Expiration date of the lot/batch: 2011-09-08
- Storage condition of test material: Stored in a desiccator at room temperature, protected from light.

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: Concentrations of the test item and control were analytically verified via standardized photometric test at the beginning and end of the test or whenever 100 % mortality occurred.
- Sampling method: All test item concentrations and the pH-control of 100 mg/L (highest test concentration) were diluted with dilution water prior to analysis. The control was analysed directly.
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C until start of analysis, if necessary.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Eluate: Dilution water
- Differential loading: 10 - 18 - 32 - 56 - 100 mg/L
- Controls: 7 fish in dilution water without test item were tested under the same test conditions as the test replicates.

Test organisms

Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebrafish
- Source: Niedersächsischer Landesbetrieb für Wasserwirtschaft, Küsten- und Naturschutz, Hildesheim, Germany
- Length at study initiation (length definition, mean, range and SD): Average body length 2.14 cm
- Weight at study initiation (mean and range, SD): Average body weight 0.11 g
- Method of breeding: Holding was performed at the test facility at 23 +/- 2 °C and diffuse light (0.1 - 10 µmol photons x m-2 x s-1, natural photoperiod). Tap water of local origin was used for holding and testing. The water was filterred on activated charcoal and aerated for at least 24 h to remove chlorine. Nominal water parameters: Total hardness: 10 - 250 mg CaCO3/L; pH-value: 6.0 - 8.5. The water is analysed biannula acc. to German tap water regulation. The water was changed at least once per week. the dissolved oxygen concentration was more than 80 % of the air saturation value.
- Feeding during test: No feeding during test



ACCLIMATION
- Acclimation period: Zebrafish with al least 12 days of acclimatisation and mortality < 5% within these days before the study starts were used.
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: 4 % of the fish body weight per feeding day. The test fish were not fed 24 h before the test started. Food: Stoer perlets, SERA GmbH, 52518 Heidelberg, Germany
- Feeding frequency: 3 times per week
- Health during acclimation (any mortality observed): No mortality observed, no disease treatments were administered thwoughout holding and testing.


Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h

Test conditions

Hardness:
Total Hardness at day 0: 60 mg/L as CaCO3
Test temperature:
Minimum Temperature [°C] 23.6
Maximum Temperature [°C] 25.9
Mean Temperature
± Standard Deviation [°C] 24.4 ± 0.534
pH:
Nominal test item concentration [mg/L] pH Value
Day 0 100 pH-control
6.97
100
3.19
56
3.83
32
5.88
18
6.54
10
7.07
Control 7.11
Day 1 100 pH-control
7.26
100
3.22
56
3.87
32
6.36
18
6.94
10
7.07
Control 7.09
Day 2 100 pH-control
7.26
100
-
56
-
32
6.82
18
7.08
10
7.24
Control 7.21
Day 3 100 pH-control
7.21
100
-
56
-
32
6.76
18
7.06
10
7.20
Control 7.10
Day 4 100 pH-control
7.30
100
-
56
-
32
6.89
18
7.08
10
7.18
Control 7.05
Dissolved oxygen:
Nominal test item concentration [mg/L] Oxygen- Saturation
[%]
Day 0 100 pH-control
100
100
100
56
100
32
100
18
100
10
100
Control 100
Day 1 100 pH-control
100
100
100
56
100
32
100
18
100
10
100
Control 100
Day 2 100 pH-control
100
100
-
56
-
32
100
18
100
10
100
Control 100
Day 3 100 pH-control
100
100
-
56
-
32
100
18
100
10
100
Control 100
Day 4 100 pH-control
100
100
-
56
-
32
100
18
100
10
100
Control 100
Salinity:
Not measured, freshwater
Nominal and measured concentrations:
Please refer to "Any other information on materials and methods"
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass aquaria
- Type (delete if not applicable): open, loosely covered by glass tops
- Material, size, headspace, fill volume: glass, 2 L test volume per vessel
- Aeration: Gentle aeration
- Renewal rate of test solution (frequency/flow rate): One application at test start
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: less than 1 g fish per L test solution


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap water, local origin, pH: 6.0 - 8.5
- Total organic carbon: 10 - 250 mg CaCO3/L
- Chlorine: The dilution water was filtered as described above
- Culture medium different from test medium: No
- Intervals of water quality measurement: The water is analysed biannual


OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: natural
- Light intensity: 0.1 - 10 µmol photons x m-2 x s-1


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Fish were considered dead if there was no visible movement (e.g. gill covers movement) and if touching of the caudal peduncle produced no reaction. Records were kept of visible abnormalities (e.g. loss of equilibrium, swimming behaviour, respiratory function, pigmentation, etc.). Observations were made after 24, 48, 72 and 96 h.


TEST CONCENTRATIONS
- Range finding study: Yes
Cumulative Mortality [%] in the Preliminary Test
(n = 5)
Test item concentration [mg/L] Test Duration [hours]
24 48 72 96
100 100 --- --- ---
10 0 0 0 0
Control 0 0 0 0
Reference substance (positive control):
no

Results and discussion

Effect concentrations
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
100 other: mg/L (with Adjustment of pH); 32 mg/L (without Adjustment)
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
- Behavioural abnormalities: Not observed
- Mortality of control: 0%
- Other adverse effects control: Not observed
- Abnormal responses: Not observed
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Not observed
Results with reference substance (positive control):
No reference substance tested
Reported statistics and error estimates:
After 24 - 96 h only 0 and 100 % mortality were found. No evaluation via regression analysis was carried out. Hence the LC50 is the geometric mean value of the concentrations with 0 and 100 % mortality. The concentration causing 0 and 100 % mortality were chosen as lower and upper confidence limit. The concentrations causing 0 and 100 % mortality (LC0 and LC100) as well as the no effect level (NOEC) after 96 h were determined directly from the test results.

Any other information on results incl. tables

               Observations in the Test Vessels

Nominal test item concentration [mg/L]

Effect*

Test Duration [h]

24

48

72

96

100 pH-control

(1)

7/7

7/7

7/7

7/7

100

(E)

7/7

---

---

---

56

(E)

7/7

---

---

---

32

(1)

7/7

7/7

7/7

7/7

18

(1)

7/7

7/7

7/7

7/7

10

(1)

7/7

7/7

7/7

7/7

Control

(1)

7/7

7/7

7/7

7/7

*) The number in brackets correspond to the following observation:

  (1)         =  behaviour            ---            =  No Observations due to 100 % mortality         

                                 (E)              =  Exitus lethalis   

Cumulative Mortality [%] in the Test Vessels

Nominal test item concentration

[mg/L]

Test Duration [h]

24

48

72

96

100 pH-control

0

0

0

0

100

100

100

100

100

56

100

100

100

100

32

0

0

0

0

18

0

0

0

0

10

0

0

0

0

Control

0

0

0

0

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In this study Phosphorpentoxid caused pH-related lethal effects at the test item concentrations of 56 and 100 mg/L whereas a concentration of 100 mg/L with adjustment of the pH-value to 6.97 did not cause any effects.
Executive summary:

The acute toxicity of the test item Phosphorpentoxid (batch no.: DEH2 018965)to fish (zebrafish) was determined according to OECD-Guideline for Testing of Chemicals No. 203 (1992) from 2009-12-14 to 2009-12-18 at Dr.U.Noack-Laboratorien, 31157 Sarstedt , Germany. A static test with 5 test concentrations 10 – 18 – 32 – 56 - 100 mg/L (factor 1.78) and for pH-control an additional replicate of the highest concentration with correction of the pH-value was performed. Duration of the test was 96 h. 7 test organisms were exposed to each test concentration and control. Water quality parameters pH-value, temperature and oxygen-saturation measured after 0, 24, 48, 72 and 96 h were determined to be within the acceptable limits. The concentrations of the test item Phosphorpentoxid were analysed via standardized photometric test at all concentration levels at test start and test end. Recovery rates of Phosphorpentoxidat the beginning of the study were in the range of 80 - 98 % and at the end in the range of 49 – 100 % of the nominal values. For details of the analytical results please refer to part 9. At the test item concentration of 56 and 100 mg/L Phosphorpentoxidcaused pH-related lethal effects whereas a concentration of 100 mg/L with adjustment of the pH-value to 6.97 did not cause any effects. All effect levels are given based on nominal concentrations of the test item Phosphorpentoxid.

LC-Values with 95 % Confidence Intervals

                                               (based on nominal test item concentrations)

Values with Adjustment of pH

Values without Adjustment of pH

Test duration [h]

LC 50

p = 95 % [mg/L]

LC 50

p = 95 % [mg/L]

24

> 100

n. a.

42

32 - 56 *

48

> 100

n. a.

42

32 - 56 *

72

> 100

n. a.

42

32 - 56 *

96

> 100

n. a.

42

32 - 56 *

LC100 =

> 100

56

Lowest test item concentration

with 100 % mortality after 96 h

LC0 =

   100

32

Highest test item concentration

with 0 % mortality after 96 h

NOEC (0-96 h) =

No observed effect concentration

   100

32